Cleaning composition containing a type II endoglycosidase

ABSTRACT

A cleaning composition is disclosed. The composition contains a first enzyme where the enzyme can be Endo-D, Endo-H, Endo-L, Endo-C, Endo-CII, Endo-F-Gal type, Endo-F and PNGaseF and a second enzyme where the enzyme can be a protease, lipase, nuclease, glycosidase, an enzyme different from the first enzyme, and any combination of these. The composition also contains a detergent surfactant and a builder. The composition can be used in a method for cleaning a surface on which is bound a glycoside-containing substance. The substance can be blood or components thereof, fecal matter or components thereof or microorganisms. The surface can be fabric, biological tissue, tooth enamel, contact lens, glass, ceramic, metal, metal alloy, plastic, plant, fruit and vegetable.

This is a division of application Ser. No. 428,361, filed Oct. 27, 1989,now U.S. Pat. No. 5,238,843.

FIELD OF THE INVENTION

The present invention relates to methods and formulations for removingglycoside-containing substances, such as glycoproteins, from surfaces bytreatment with Type II endoglycosidase alone or in combination withother enzymes and/or detergents.

BACKGROUND OF THE INVENTION

The use of enzymes to remove stains comprising proteins and/orcarbohydrates, in combination with various detergents, is well known inthe art of detergent formulations. Such enzyme formulations are designedto remove various types of stains from soft surfaces such as cloth andhard surfaces such as porcelain and metal. Thus, for example, proteasessuch as trypsin, pancreatin, papain and bromelain have reportedly beenused in detergent formulations to remove proteinaceous stains withvariable degrees of success. Specific glycosidases such as cellulase,lysozyme, amylase and glucanase, on the other hand, have been formulatedwith various detergents for removal of certain carbohydrate stains.Other detergent formulations have combined proteases and glycosidasesfor stain treatment.

Some of the glycosidases used in detergent formulations, e.g.,β-amylase, α-galactosidase and β-galactosidase, are exoglycosidaseswhich cleave one or more terminal residues from an oligosaccharide orpolysaccharide. Other glycosidases, e.g. cellulase and α-amylase areendoglycosidases which are reactive with specific internal linkageswithin an oligo- or polysaccharide substrate. Such endoglycosidases arereferred to herein as Type I endoglycosidases. Although formulations ofdetergent with one or more proteases and/or glycosidases (including TypeI endoglycosidases) have greatly improved stain removal, many stains,e.g. blood, fecal material and body soil stains, often leave a residualstain after treatment.

In the art of contact lens cleaning, similar enzyme/detergentformulations have been used to clean and sterilize hard and soft contactlenses. In many cases, these formulations have been used to degrade thebiofilm which forms on the surface of contact lenses and which is usedby various ophthalmic pathogens such as Pseudomonas aeruginosa andStaphylococcus epidermidis to adhere to such lens. See, e.g., Duran, J.A., et al. (1987), Arch. Ophthalmol, 105 106-109; Stern, G. A., et al.(1987), Ophthalmology, 94, 115-119 (which reports the treatment of mucincoated contact lenses with various enzymes such as pancreatin, papain,trypsin and neuraminidase to inhibit Pseudomonas adherence); andSlucher, M. M., et al. (1987), Arch. Ophthalmol, 105, 110-115.

The use of biofilms for microbial adhesion is not limited to contactlenses. Thus, Streptococcus mutans reportedly uses extracellularpolysaccharides to adhere to tooth enamel. EPO Publication No. 0195672reports the use of α-1,3 glucanase or α-1,6 glucanase to cleave theextracellular polysaccharides used by Streptococcus mutans to adhere totooth enamel.

The effect of certain enzymes on cells adhered to glass surfaces hasalso been reported by Danielsson, A., et al. (1977), Botanica Marina,20, 13-17. As reported therein, Pseudomonas species isolated from seawater was adhered to glass slides. Thereafter, the slides were treatedwith either pronase, trypsin, α-amylase (a Type I endoglycosidase), orlysozyme (also a Type I endoglycosidase). In this report, treatment withthe proteolytic enzymes pronase and trypsin resulted in the release of aportion of the population of adhered bacteria, whereas the celldegradative enzyme lysozyme showed diminished activity compared to theproteolytic enzymes. The α-amylase reportedly had no effect at all. Inaddition to the attachment of microorganisms to contact lenses, toothenamel and glass surfaces, many other surfaces are subject to microbialattachment. See, e.g., Marrie, T. J., et al. (1984), J. Clin.Microbiology, 19, 991-914 (bacterial attachment to cardiac pacemakerleads and powerpacks); Freimer, N. B., et al. (1978), Acta. Path.Microbiol. Scand. Sectb., 86, 53-57 (binding of microorganisms tomacrophages); and Mirelman, et al. (1982), Tokai J. Exp. Clin. Med., 7,77-183 (microbial adherence to mammalian mucosal surfaces). Variousmechanisms have been proposed to describe the adhesion ofmicroorganisms, such as bacteria, to non-biological solid surfaces. See,e.g. Fletcher, M. (1987), Microbiological Sciences, 4, 133-136, andDuddridge, J. E., et al. (1983), Factors Affecting the Adhesion ofBacteria to Surfaces in Microbial Corrosion, Delco Printing Co., Ltd.,pp. 28-35. Although these references discuss microbial adherence tovarious surfaces and the factors which may be involved in suchattachment, they do not discuss the control of microorganism growth onsuch surfaces or their removal therefrom.

Type II endoglycosidases, as used herein, are a category ofendoglycosidases which are capable of cleaving specific internalglycosidic linkages found in glycoproteins. These endoglycosidasescleave all or part of the carbohydrate moiety from a glycoproteindepending on the location of the reactive glycosidic linkage in theglycoprotein. Examples include endo-β-N-acetylglucosaminidases (Endo-D,Endo-H, Endo-L, Endo-CI, Endo-CII, Endo-F-Gal type and Endo-F),endo-α-N-acetylgalactosaminidase and endo-β-N-galactosidases. See, e.g.Tarentino, A. L., et al. (1985), Biochem, 24, 4665-4671; Arakawa, M., etal. (1974), J. Biochem., 76, 307-317; Plummer, T. H., et al. (1984), J.Biochem, 259, 10700-10704; Tarentino, A. L., et al. (1975), Biochem. andBiophys. Res. Comm., 67, 455-462; and Trimble, R. B., et al. (1984),Anal. Biochem., 141, 515-522; and "Glycoprotein and ProteoglycanTechniques" (1985) by J. G. Beeley, Chapter 6, pp. 153-300, Elsevier,Amsterdam, N.Y., Oxford. In addition to having a specificity for theinternal glycosidic linkages of glycoproteins, at least oneendoglycosidase (endo-β-N-acetylglucosaminidase H) has also demonstrateda specificity which produces the cleavage of lipid-linkedoligosaccharides (Chalifour, R. J., et al. (1983), Archives of Biochem.and Biophys., 229, 386-394) and reportedly di-N-acetylchitobioselinkages in oligosaccharides and glycoproteins (Tarention, A. L., et al.(1974), J. Biol. Chem., 249, 811-817).

Such Type II endoglycosidases, in general, have been used primarily foranalytical purposes, e.g. the determination of protein or carbohydratesequence and/or the structure and function of specific glycoproteins.See, e.g. Hsieh, P., et al. (1982), J. Biolchem., 258, 2555-2561, andGeyar, R., et al. (1984), Eur. J. Biochem., 143, 531-539. In a recentreport, a Type II endoglycosidase was reportedly used to analyze aglycoprotein antigen from Leishmania mexicana amazonensis. Chin ShenChang, et al. (1986), Mol. Biochem. Parasitol 18, 197-210. Thisglycoprotein antigen was first immunologically bound to immunobeads.After reacting the immunologically bound glycoprotein with analyticalamounts of Endo-H, the immunobeads were washed and boiled in buffercontaining 1% SDS in preparation for polyacrylamide gel electrophoresis.This analysis revealed a decrease in molecular weight attributed to thecleavage of carbohydrate from the immunologically bound glycoproteinantigen.

Type II endoglycosidases, however, have not been used to removesubstances, including glycoproteins and glycolipids, from surfaces ofsubstances such as fabric, contact lenses, metals, ceramics, cells,tissue and the like. Nor have they been used to control microorganismgrowth in suspension or on such surfaces.

Glycosidases have been used in combination with other enzymes forremoval of various materials. β-glycosidases are described ascarbohydrate-metabolizing enzymes in Anderson, et al. (1964), Biochem.J., 90, 30. Neuraminidase (N-acetylneuraminiate glycohydrolase)inhibitors are viewed as possible anti-viral, antibacterial agents inKhorlin, et al. (1979), FEBS Letters, 8, 17; and Haskell, et al. (1970),J. Med. Chem,, 13, 48. Dextranase is described as catalyzing hydrolysisof bacterial polysaccharide, dextran (α-1,6-glucan), to isomaltoseresidues in Chalet, et al. (1970), Appl. Microbiol., 20, 421. Lysozyme(muramidase) is described as hydrolyzing glycosidic linkages in themucopolysaccharide cell wall structure of a variety of microbes inChipman, et al. (1969), Science, 165, 454 and Montague (1964), Biochem.Biophys. Acta., 86, 588. Lastly, inhibition of lysozyme by D-glucosaminederivatives is described in Neuberger, et al. (1967), Nature, 215, 524.

Type II endoglycosidases such as endo-β-N-acetylglucosaminidase H, D, Fand/or PNGase F have not, however, previously been combined withantimicrobial agents to form antimicrobial compositions.

The references discussed above are provided solely for their disclosureprior to the filing date of the instant case, and nothing herein is tobe construed as an admission that such references are prior art or thatthe inventors are not entitled to antedate such disclosure by virtue ofprior invention or priority based on earlier filed applications.

SUMMARY OF THE INVENTION

It is an object herein to provide methods utilizing Type IIendoglycosidases alone or in combination with other enzymes, detergents,surfactants and/or disulfide cleaving reagents to facilitate the removalof substances including glycoproteins and glycolipids from materialssuch as fabric, contact lenses, metallic surfaces, plastic surfaces,ceramic surfaces, cell surfaces, tissue and the like.

It is a further object herein to provide formulations containing Type IIendoglycosidases useful in practicing such methods.

In accordance with the objects of the invention, methods are providedfor removing at least a portion of a substance from a surface to whichit is bound, other than by an immunological bond. The substance has aproximal portion bound to a surface, a distal portion extendingoutwardly from the proximal portion, and a linkage disposed at thejuncture of the proximal and distal portions which is reactive with aType II endoglycosidase. The method comprises contacting the boundsubstance with a Type II endoglycosidase to cleave the distal portion ofthe substance from the proximal portion thereby releasing the distalportion from the surface. If such releasing does not remove the distalportion from the surface, such removal may be facilitated by contactingthe distal portion with a washing solution, a detergent formulationincluding those with surfactants or a fluid containing a second enzymesuch as a different glycosidase, protease, lipase, nuclease orcombinations thereof.

The invention further includes a method for releasing at least a portionof a glycoside-containing substance from a surface to which it is boundby contacting the glycoside-containing substance with at least a firstand a second enzyme to release a cleaved portion of theglycoside-containing substance from the surface. The first enzyme is aType II endoglycosidase reactive with a glycosidic linkage in theglycoside portion of the substance. The second enzyme is reactive with asecond linkage in the glycoside-containing substance other than theglycosidic linkage with which the first enzyme is reactive. Such secondenzymes may include glycosidases (including a second Type IIendoglycosidase), proteases, lipases and combinations thereof. Theremoval of a portion of the glycoside-containing substance may befacilitated by contacting the cleaved glycoside-containing substancewith a detergent including those containing a surfactant.

The invention also includes a method for releasing from a surface atleast a portion of a glycoside-containing substance having acarbohydrate portion, an aglycon portion and a glycosidic linkage at thejuncture of the carbohydrate and aglycon portions wherein the glycosidecontaining substance is bound to a surface through the carbohydrateportion. The method comprises cleaving the glycosidic linkage with aType II endoglycosidase to release the aglycon portion of theglycoside-containing substance from the surface. Removal of the cleavedportion of the glycoside-containing substance may, if necessary, befacilitated by treatment with a second enzyme, detergent and/orsurfactant.

In each of the above methods, a disulfide cleaving reagent may beemployed to denature protein associated with the glycoside-containingsubstance thereby facilitating cleavage by the Type II endoglycoside orsecond enzyme or removal by detergent and/or surfactant.

The invention also includes formulations. One composition comprises atleast a first and a second enzyme wherein the first enzyme is a Type IIendoglycosidase. The second enzyme is selected from the group of enzymesconsisting of proteases, glycosidases, lipases and combinations thereof.Such formulations may also include detergents and/or surfactants as wellas disulfide cleaving reagents.

Another composition includes Type II endoglycosidase and detergentand/or surfactant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the common core structure of N-linked and O-linkedglycoproteins.

FIG. 2 depicts the substrates and known cleavage sites for various TypeII endoglycosidases.

FIG. 3 is a generic presentation of the protein amino acids,carbohydrate residues and cleavage sites of FIG. 2.

FIG. 4 depicts the core structure of an N-linked glycoprotein, thecleavage site of a Type II endoglycosidase and the relationship betweenthe protein and carbohydrate units and the aglycon and carbohydrateportions produced upon cleavage with a Type II endoglycosidase.

FIGS. 5A-5E depict various mechanisms whereby a glycoside-containingsubstance, microorganisms or substances reactive with Type IIendoglycosidase may be released from a surface by treatment with Type IIendoglycosidase alone or in combination with a second enzyme.

FIGS. 6A and 6B are electron micrographs (8100×) of nylon swatchesstained with fecal matter and treated either with or without Endo-H.

FIGS. 7A through 7H are electron micrographs (5000×) showing the effectof Endo-H and other carbohydrase enzymes on cotton swatches stained withfecal matter.

FIGS. 8, 9 10A and 10B demonstrate the effect of various concentrationsof Endo-H and chlorhexidine, alone or in combination, on the viabilityof E. coli.

FIGS. 11 and 12 demonstrate that a detergent composition containingEndo-H is more effective in the removal of S. aureaus from swine skinthan a detergent composition not containing Endo-H.

FIG. 13 demonstrates that Endo-H is more effective in removing mold froma shower curtain than water or a detergent composition. The centerphotograph is of a portion of the shower curtain. The other fourphotographs are enlargements of the corresponding quadrants of thecenter photograph.

FIG. 14 demonstrates the antimicrobial effect of Endo-H in combinationwith different antimicrobial agents.

FIGS. 15A-B and 16A-B demonstrate the effect of Endo-H on differentspecies of yeast.

FIGS. 17 and 18 demonstrate the enhanced removal of fecal matter fromdiaper material by a detergent composition containing Endo-H.

DETAILED DESCRIPTION OF THE INVENTION

Type II endoglycosidases and formulations employing suchendoglycosidases are used in the methods of the present invention torelease and/or remove substances reactive with Type II endoglycosidasesfrom a surface. The mechanism of this reactivity is not known withcertainty. In some cases, such substances are glycosides orglycoside-containing substances which are believed to have glycosidiclinkages that are known cleavage sites for Type II endoglycosidases orlinkages which are closely related to such cleavage sites.

As used herein, "Type II endoglycosidases" are enzymes which are capableof cleaving linkages at or near the juncture of the protein andcarbohydrate units of a glycoprotein. Preferably, such Type IIendoglycosidases are capable of cleaving at least one glycosidic linkagewithin about three glycosidic linkages of the protein-carbohydrate unitjuncture (including the glycosidic linkage comprising theprotein-carbohydrate junction). Most preferably, such glycosidiclinkages are within about two glycosidic linkages of theprotein-carbohydrate unit juncture (see FIGS. 1, 2 and 3).

Type II endoglycosidases are also defined by their specificities for theparticular glycosidic linkages shown in FIG. 1 for the known corestructures of N-and O-linked glycoproteins. These correspond to theglycosidic linkages between the amino acids serine, threonine orasparagine and the first carbohydrate residue and the glycosidiclinkages between at least the first, second and third carbohydrateresidues. Although this core structure will be described in more detailhereinafter in terms of the specific glycosidic linkages which exist inknown core structures, such specific linkages are not to be construed aslimiting to this definition of Type II endoglycosidases. Accordingly,all possible glycosidic linkages between these amino acids andcarbohydrate residues define the core structure of N-and O-linkedglycoprotein used to identify Type II endoglycosidases.

Type II endoglycosidases are not limited by the present knowledge of theglycoprotein core structure and the specificity of knownendoglycosidases for such core structures. A comparison of the corestructures in FIG. 1 with the known substrates for Type IIendoglycosidases in FIG. 2 indicates that Type II endoglycosidases foreach of the possible cleavage sites in the core structures in FIG. 1, ifthey exist, have not yet been identified. Moreover, other corestructures may also exist which have not yet been identified.Endoglycosidases reactive with linkages in such, as yet, unknown corestructures are also Type II endoglycosidases. Accordingly, theglycosidic linkages in glycoproteins which define Type IIendoglycosidases are not limited to those located within the first threeglycosidic linkages closest to the protein unit of the glycoprotein butmay extend to more distant glycosidic linkages in the core structure,e.g. to the fourth or fifth glycosidic linkage from the protein unitdepending on the core structure identified.

The specificity for the core structure of glycoproteins provides aconvenient definition of Type II endoglycosidases which distinguishesthem from Type I endoglycosidases. Type I endoglycosidases cleavespecific linkages in oligo- or polysaccharides but generally are notreactive with those core structure glycosidic linkages in glycoproteinswhich define Type II endoglycosidases. Examples of Type Iendoglycosidases and the linkages with which they are reactive are shownin Table I.

                  TABLE I                                                         ______________________________________                                        Type I       Substrate                                                        Endoglycosidase                                                                            oligo-or polysaccharide                                          ______________________________________                                        α-amylase                                                                            α1-4                                                                    (Glc-Glc).sub.n                                                               ↑                                                          cellulase    β1-4                                                                     (Glc-Glc).sub.n                                                               ↑                                                          hyaluronidase                                                                              β1-3 β1-4 β1-3                                                 GlcA-GlcNAc-GlcA-GlcNAc                                                       * ↑                                                        lysozymes:   β1-4 β1-4 β1-4                                    hen egg white                                                                              GlcNAc-MurNAc-GlcNAc-MurNAc                                      lysozyme     ↑ *                                                        T4 lysozyme                                                                   mutanolysin                                                                   pullulanase  α1-4 α1-4 α1-6 α1-4                                   Glc-(Glc-Glc-)Glc-Glc                                                         ↑                                                          ______________________________________                                         *GlcA is DGlucuronic Acid                                                     MurNAc is NAcetylmuramic Acid                                                 ↑ Indicates cleavage site.                                         

Specific glycosidic linkages in glycoproteins which define Type IIendoglycosidases and which identify preferred Type II endoglycosidasesare shown in FIG. 2. The cleavage sites are identified by a verticalarrow. A generic presentation of the protein amino acids, carbohydrateresidues and cleavage sites of FIG. 2 is shown in FIG. 3. As can beseen, Type II endoglycosidases preferably cleave the first, second orthird glycosidic linkages in N- or O-linked glycoproteins. Theselinkages comprise the glycosidic linkages (1) between asparagine, serineor threonine in the protein unit and the first carbohydrate residue, (2)between carbohydrate residues 1 and 2 and (3) between carbohydrateresidues 2 and 3, respectively. This specificity is defined primarily bythe carbohydrate sequence of the glycoprotein with specificity andreactivity being influenced to some extent by the protein unit of theglycoprotein. Thus, with regard to glycosidic linkages 2 and 3(comprising glycosidic linkages between carbohydrate residues only),Type II endoglycosidases may be reactive with identical or similarglycosidic linkages located in other regions of a glycoprotein, perhapsquite distant from the juncture of the protein and carbohydrate units ofthe glycoprotein.

An application of the above definition to a particular glycoprotein isillustrative. Bovine thyroglobulin has been analyzed usingendo-β-N-acetylglucosaminidase-H (Endo-H), α-mannosidase andβ-mannosidase. Tarentino, A. L. et al. (1973) J. Biol. Chem., 218, 5547.The Endo-H hydrolyzed the glycosidic linkage between the two N-acetylD-glucosamines, one of which was N-linked to an asparagine in theprotein unit of the thyroglobulin. The oligosaccharide or carbohydrateportion of the thyroglobulin released upon treatment with Endo-H wasalso treated with α- and β-mannosidase. Since neither of these enzymeshas a specificity for the substrates corresponding to those shown inFIGS. 1, 2 or 3, they are not Type II endoglycosidases and can becharacterized as either an exoglycosidase or Type I endoglycosidase. Thespecificity of the Endo-H is the same as that shown for Endo-H in FIG. 2and Endo-H is therefore a Type II endoglycosidase. This is of course atrivial application. But if a new endoglycosidase (e.g. Endo-X) isdiscovered which also demonstrates this specificity or one or more ofthe other specificities in FIGS. 1, 2 or 3, that Endo-X would also be aType II endoglycosidase.

This definition of a Type II endoglycosidase based on its specificityfor glycoproteins, however, should not be construed as a limitation onthe mechanism utilized by Type II endoglycosidases to release and/orremove a substance from a surface. Although it will be assumed in someinstances that Type II endoglycosidases cleave at least a part of aglycoside from a surface by reacting with a glycosidic linkage in theglycoside, the invention is not limited to such cleavage. Rather, theaction of Type II endoglycosidases is defined functionally by theirability to cleave from a surface at least a part of any substancereactive with a Type II endoglycosidase.

As used herein, the term "endoglycosidase" comprises Type I and Type IIendoglycosidases.

As used herein, "glycoside" refers to a polymer which has one or more"carbohydrate portions" covalently attached through a glycosidic linkageto an "aglycon portion". This definition of glycoside is derived fromthe common definition of glycoside which refers to a compound thatyields on hydrolysis a sugar and an aglycon, the aglycon being thenon-sugar compound resulting from such hydrolysis. As used herein, aglycoside produces an aglycon and an oligo- or polysaccharidecarbohydrate portion when cleaved by a Type II endoglycosidase. Theaglycon unit, however, is not limited to a non-sugar compound since TypeII endoglycosidases may hydrolyze a glycoside to produce an aglyconportion containing one or more sugar residues depending on the cleavagesite of the Type II endoglycosidase. Further, the aglycon portion may bequite complex as might be the case with peptidoglycans where crosslinkedpeptides can be found attached to a matrix of carbohydrate. Thus,glycosides include glycoproteins, glycolipids, peptidoglycans and thelike which upon treatment with a Type II endoglycosidase produce acarbohydrate portion and aglycon portion wherein the carbohydrateportion and aglycon portion are defined by the cleavage site of the TypeII endoglycosidase. This definition of glycoside will be apparent fromthe discussion which follows.

As used herein, "glycoprotein" refers to a glycoside which has one ormore oligo- or polysaccharides covalently attached to a peptide orprotein. Oligo-and polysaccharides are sometimes referred to herein as"carbohydrate units". Such carbohydrate units, however, may be differentfrom the "carbohydrate portion" of a glycoside. As shown in FIG. 4, acarbohydrate unit comprises the entire oligo- or polysaccharide attachedto a second class of molecule, e.g., to a protein or peptide as in aglycoprotein or to a lipid as in a glycolipid. If the Type IIendoglycosidase cleaves the carbohydrate unit at its juncture with, forexample, a protein then the carbohydrate unit is synonymous with thecarbohydrate portion of a glycoside. If, however, the Type IIendoglycosidase cleaves the carbohydrate unit at a glycosidic linkagewithin the carbohydrate unit, then the carbohydrate portion of theglycoside formed by such cleavage will be less than the entirecarbohydrate unit. This difference is shown in FIG. 4 for a Type IIendoglycosidase cleavage site indicated by the arrow.

The carbohydrate units of a glycoprotein may be oligosaccharidescontaining 1 to 10 carbohydrate (sugar) residues or shortpolysaccharides which usually contain between 10 to 25 carbohydrateresidues. Many glycoproteins are produced by higher organisms such aseukaryotes including yeast and mammalian cells. The linkage between thecarbohydrate unit and the peptide or protein unit of a glycoprotein is aglycosidic linkage which results from a condensation reaction between anamino acid side chain of the protein unit and the anomeric carbon on thefirst residue of the carbohydrate unit. Such glycosidic linkages inmammalian glycoproteins are either N-glycosidic linkages (carbohydratelinked to the amido nitrogen of asparagine) or O-glycosidic linkages(carbohydrate linked to the hydroxy oxygen of serine or threonine).

The carbohydrate residues (monosaccharides) of a carbohydrate unit(oligo or polysaccharide) may be joined together in many different ways.Thus, such carbohydrate units may be unbranched, linear structures ormay be complex branched structures. In general, however, each of thecarbohydrate residues in the carbohydrate unit is linked by way of aglycosidic linkage wherein the anomeric carbon of one carbohydrateresidue is condensed with the hydroxyl carbon in another carbohydrateresidue. Such glycosidic bonds may be either alpha or beta depending onthe configuration of the anomeric carbon. The anomeric carbon of oneresidue may be combined with any of the hydroxyl carbons in anothercarbohydrate residue. Thus, the complexity of many glycoproteins arisesfrom the many different glycosidic linkages which are found in thecarbohydrate units of such molecules.

Many membrane glycoproteins carry asparagine-linked carbohydrate units(carbohydrate units linked to asparagine in a peptide via anN-glycosidic linkage). The structure of such asparagine linkedglycoproteins can be quite complex. See e.g., Schachterh (1984) ClinicalBiochemistry 17, 3-14. The structure of many of these asparagine linkedmembranous glycoproteins from a variety of sources (e.g., erythrocyteplasma membrane glycoproteins, viral envelope glycoproteins) as well asthe structure of non-membranous soluble glycoproteins indicate that thetwo types of glycoproteins share many structural features. Id. at 3. Thecommon core structure of such asparagine-linked glycoproteins is shownin FIGS. 1 and 4, wherein GlcNAC is N-acetyl D-glucosamine and Man ismannose. The α1-6, α1-3 and β1-4 designations describe the type ofglycosidic linkage between the various carbohydrate residues. This corelinkage forms the basis of numerous glycoproteins having any of a numberof carbohydrate residues attached to the core. Id. at 5.

O-linked glycoproteins contain a core structure wherein the protein unitof the glycoprotein is coupled to the carbohydrate unit through thehydroxyl group of either serine or threonine. A common feature of thiscore structure is the presence of N-acetyl D-galactosamine (GalNAc)linked to serine or threonine. Other details of such glycoproteins areshown in FIG. 1 where NeuAc is N-acetylneuraminic acid, Gal is Galactoseand L-Fuc is L-Fucose. When Gal is the second carbohydrate residue theglycosidic linkage between GalNAc and Gal is usually β1-3. For review ofthe structure biosynthesis and function of glycoproteins including N-and O-linked glycoprotein, see Berger E. G. et al. (1982) Experimentia,38, 1229-1258.

Lower organisms such as prokaryotes, e.g., the bacteria E. coli,Pseudomonas species, Bacillis species and the like, producepeptidoglycans rather than glycoproteins. Peptidoglycans are found inbacterial cell walls and typically have a polysaccharide backbone ofalternating N-acetylglucosamine and N-acetylmuramic acids. Peptide sidechains are sometimes associated with the N-acetylmuramic acid residueswith cross-linked peptide bridges often being interposed between thepeptide side chains. The cell wall of Gram-positive bacteria typicallycomprises approximately 10% peptidoglycan whereas the cell wall ofGram-negative bacteria typically have a peptidoglycan content of about50%.

Peptidoglycans, however, are not glycoproteins, at least to the extentthat specific glycosidic linkages in glycoproteins are used to definethe class of Type II endoglycosidase. Thus, Endo-H is a Type IIendoglycosidase because it cleaves the glycosidic linkage between thetwo N-acetylglucosamine sugar residues found in some glycoproteinscontaining N-linked oligosaccharides. See FIG. 4. Endo-H, however, mayalso have an as yet undefined reactivity with peptidoglycan since it iscapable of facilitating the removal of fecal matter from a surface suchas cloth swatches. Such fecal matter is known to contain peptidoglycansassociated with intestinal bacteria. Lysozymes are enzymes which arereactive with peptidoglycan. Lysozymes, such as hen egg white lysozyme,T4 lysozyme and mutanolysin (Goodman, et al. (1981), J. Bacteriol, 146,755), however, are not Type II endoglycosidases. This is because they donot have a substantial reactivity with the unique glycosidic linkagesfound in N- and O-linked glycoproteins used to define Type IIendoglycosidases. They are, however, reactive with peptidoglycans toproduce disaccharides of N-acetylglucosamine and N-acetylmuramic acidcontaining attached peptide side-chains. As such, lysozymes are moreappropriately characterized as a Type I endoglycosidase. Thus, eventhough lysozymes and Endo-H may have an overlap in reactivity withpeptidoglycans, they are mutually exclusive, for the most part, withregard to Endo-H's specificity for, and lysozyme's substantial lack ofreactivity with, the glycosidic linkages in glycoproteins which defineType II endoglycosidases.

As used herein, a "glycoside containing substance" or "glycoproteincontaining substance" is a glycoside or glycoprotein alone or aglycoside or glycoprotein combined with another component. Thus,glycoside-containing substances include glycosides such as glycoproteinenzymes, e.g., alkaline phosphatase, bromelain, carboxypeptidase-Y;glycoprotein hormones, e.g., chorionic gonadotropin, erythropoietin;lectins, e.g., those derived from potato and soybean; serumglycoproteins, e.g., IgG immunoglobulin, thyroglobulin, prothrombin andthe like and miscellaneous glycoproteins such as hemoglobin andinterferon; and complex carbohydrates. Examples of glycosides combinedwith another component include glycoproteins comprising membraneconstituents, e.g., glycophorin contained by human erythrocytes,hemagglutinin contained by influenza virus, rhodopsin contained inbovine retina and collagen contained by fibroblasts. Furtherglycoside-containing substances include viral envelope glycoproteins andfecal matter which contains in part peptidoglycans associated withintestinal bacteria. Thus, viruses, fibroblasts, fecal matter etc. areconsidered glycoside-containing substances.

As used herein, a "microorganism" (sometimes referred to as aglycoside-containing microorganism) is one capable of being cleaved fromthe surface of a substance to which it is bound by a Type IIendoglycosidase. Examples include the intestinal bacteria found in fecalmatter and bacteria commonly contaminating contact lens. Other examplesinclude fungi and algae which can be cleaved from a surface by Type IIendoglycosidase.

As used herein, the term "in vitro" refers to the environment in whichthe processes and methods of the invention are practiced. It is usedonly to distinguish from the term "in vivo" which describes theenvironment in which Type II endoglycosidases are found naturally, e.g.within organisms which naturally produce Type II endoglycosidase.Accordingly, an in vitro method employing a Type II endoglycosidase is amethod or process which does not occur in nature. The term in vitro,however, is not to be construed as a limitation of such methods to "inglass" or to exclude such methods from being practiced on or in a livingorganism. The methods of the invention may be practiced on a variety ofsurfaces other than glass including fabric, contact lenses, metallicsurfaces, ceramic surfaces, cell surfaces, plastic surfaces, tissue andthe like. Further, such in vitro methods may be practiced for example inthe human oral cavity as described in more detail hereinafter.

Some known Type II endoglycosidases are listed in Table II together withthe natural biological source of such enzymes. The cleavage sites forsome Type II endoglycosidases are shown in FIG. 2. See "Glycoprotein andProteoglycan Techniques" (1985) by J. G. Beeley, Chapter 6, pp. 153-300,Elsevier, Amsterdam, N.Y., Oxford. A Type II endoglycosidase not listedin Table II is Glycopeptidase F also sometimes referred to as PNGase F.PNGase F may be obtained from Flavobacterium meningosepticum. It is alsocommercially available from Boehringer Mannheim Biochemical,Indianapolis, Ind.

                                      TABLE II                                    __________________________________________________________________________    Enzyme      Source      Typical substrate                                     __________________________________________________________________________    endo-β-N-                                                                Acetylglycosaminidases                                                        D           Diplococcus pneumoniae                                                                    N-linked complex type                                                         (peripheral sugars removed)                           H           Streptomyces plicatus                                                                     N-linked hign-mannose                                             (Streptomyces griseus)                                                                    and hybrid types                                      L           Streptomyces plicatus                                                                     N-linked low mol. wt. only                            C.sub.I     Clostridium perfringens                                                                   N-linked complex type                                                         (peripheral sugars removed)                           C.sub.II    Clostridium perfringens                                                                   N-linked high-mannose type                            F-Gal type  Sporotricum N-linked complex type                                             dimorphosphosphorum                                                                       (biantennary only,                                                            requires terminal Gal)                                F           Flavobacterium                                                                            N-linked high-mannose and                                         meminogosepticum                                                                          complex types                                         endo-α-N-                                                                           Diplococcus pneumoniae                                                                    O-linked, only                                        Acetylgalactosaminidase Gal-α1-3GalNAcl-                                endo-β-N-Galactosidases                                                              Diplococcus pneumoniae                                                                    Blood group A and B                                                           determinants                                                      Escherichia freundii                                                                      Keratan sulphate and oligo-                                       Flavobacterium                                                                            saccharides containing                                            keratolyticus                                                                             sequence R GlcNAc-β1-                                                    3Gal-β1-4GlcNAc (or Glc)                         __________________________________________________________________________

As can be seen, Endo-H, F, D, CI and Endo-F-Gal type all cleave thesecond glycosidic linkage in a glycoprotein. In the case of Endo-F-Galtype, this glycosidic linkage is between GlcNAc and Gal. For Endo-H, F,D, and CI, the cleavage is between two residues comprising GlcNAc, withspecificity being defined by the substituents U, V, W, X, Y, and Z.

Endo-H cleaves N-linked glycoproteins having a high mannose content.Thus in FIG. 2, W comprises 2-150 mannose residues, Y comprises 1-2mannose residues and X,Z,V and U are H (hydrogen). Endo-H also cleaveshybrid structures wherein W comprises 1-2 mannose residues and Y and/orZ comprise NeuNAc-Gal-GlcNAc or similar structures and V comprises H orGlcNAc. Endo-H is the preferred Type II endoglycosidase used in theformulations and methods of the invention.

Endo-D and Endo-C_(I) have similar reactivities although these enzymesare derived from different sources. Endo-D and Endo-C_(I) are active onN-linked oligosaccharides of glycoproteins and cleave a high mannosestructure containing more than a 5-mannose carbohydrate residue in whichcase X comprises mannose linked by way of an α1-3 glycosidic bond to thecore structure, W comprises mannose linked by way of an α1-6 glycosidicbond to the core structure and the remaining substituents are H in FIG.2. Endo-D also cleaves a core portion of a complex or hybrid structureafter removal of most antennary residues with exoglycosidases, in whichcase Y comprises H or GlcNAc and U comprises H or fucose in FIG. 2.

The endoglycosidase Endo-F is active on N-linked glycoproteins having ahigh mannose content wherein in FIG. 2 X and Y are one or more mannoseresidues and the remaining substituents are H. Endo-F also cleavesbiantennary hybrid structures wherein X and W comprise mannose linked tothe core structure by way of α1-3 and α1-6 glycosidic linkages and Ycomprises NeuNAc-Gal-GlcNAc or similar structure and U comprises H orfucose. Biantennary complex structures are also cleaved by Endo-F. Suchstructures comprise the substrate core structure for Endo-F in FIG. 2wherein X and Y comprise NeuNAc-Gal-GlcNAc or similar structures and Ucomprises H or fucose.

Endo-L has a similar reactivity in cleaving the second glycosidiclinkage in N-linked glycoproteins. It is specific for low molecularweight substrates comprising Man-GlcNAc-GlcNAc-Asn. Endo-C_(II)demonstrates a specificity similar to Endo H. Endo-α-N-acetylgalactosaminidase hydrolyzes glycoprotein containing oligosaccharidesO-linked to serine or threorine where GlcNAc and Gal are the first twocarbohydrate residues. The specificity of endo-β-N-galactosidase is alsoshown in FIG. 2 wherein R1 may be one of the mannoses from whichantennas in the carbohydrate unit may be formed.

The Type II glycosidase glycopeptidase F (PNGase F) cleaves the firstglycosidic linkage in N-linked glycoproteins between asparagine andGlcNAc. It cleaves high mannose structures wherein W, X and Y compriseone or more mannose residues and V and Z comprise H with fucose beingabsent from the first carbohydrate residue GlcNAc. It also cleaveshybrid structures wherein W and X comprise mannose, Y and/or Z compriseNeuNAc-Gal-GlcNAc or similar structure, V comprises H or GlcNAc withfucose typically being absent from the first carbohydrate residue.Complex structures are also cleaved by glycopeptidase F. Such structurescomprise the core structure shown in FIG. 2 wherein Y and W compriseNeuNAc-Gal-GlcNAc or similar structure, X and Z comprise H,NeuNAc-Gal-GlcNAc or similar structure, V comprises H or GlcNAc andfucose is sometimes present on the first carbohydrate residue GlcNAc.

Endo-β-N-galactosidase is known to cleave glycosydic linkages withinoligosaccharides on a glycoprotein or glycolipid. A typical glycoproteinsubstrate together with the cleavage site for Endo-β-N-galactosidase isshown in FIG. 2 where R₂ is protein, lipid or carbohydrate, and R₁ is asugar residue or hydrogen.

Of course, the invention is not limited by the present known specificityof endoglycosidases. Until recently, the endoglycosidases which havebeen commercially available have been expensive due to their relativelylow levels of expression in their naturally occurring sources.Accordingly, the reactivity of such enzymes has not been broadlyinvestigated. However, with the advent of molecular cloning, greateramounts of endoglycosidase have been or will be made available. To theextent that alternate reactivity and specificity may be discovered forthese or other endoglycosidases, such reactivity is intended to bewithin the scope of the invention.

Accordingly, as used herein, a "Type II endoglycosidase-reactivesubstance" (also referred to as a "Type II-reactive substance" or asubstance containing a "Type II reactive linkage") is any substancewhich is reactive with a Type II endoglycosidase. Included within TypeII reactive substances, of course, are glycoside-containing substancesand glycoprotein. Also included, however, are (1) other, as yet, unknownsubstrates reactive with Type II endoglycosidase at other than aglycosidic linkage, and (2) multicomponent aggregates containingcomponents having Type II reactive linkages.

For example, microorganisms, such as bacteria, can be removed fromsurfaces by treatment with Endo-H. It is presently not known how thisresult occurs. Bacteria are not known to contain linkages which arenormally reactive with Endo-H and the details of their attachment tosurfaces, other microorganisms and other substances is not wellunderstood. Yet, bacteria removal by Endo-H has been observed.

Further, other stains may involve complex aggregates of substances someof which or all of which are reactive with Type II endoglycosidase. Theterm Type II reactive substance covers all such situations. Thus, usesof Type II endoglycosidase include (1) cleaning surfaces containing TypeII-reactive substances, (2) treating Type II-reactive substances toprevent attachment to a surface, and (3) treating Type II-reactivesubstances such as microorganisms to produce an antimicrobial effect.

The Type II endoglycosidases used in the invention can be obtained fromthe organisms listed in Table II according to methods known to thoseskilled in the art. Some of the Type II endoglycosidases in Table II,e.g., Endo-H from Streptomyces plicatus (initially classified asStreptomyces griseus) and produced in S. plicatus or S. lividans andEndo-D from Diplococcus pneumoniae, are commercially available fromBoehringer Mannheim Biochemical, Indianapolis, Ind. Besides thecommercially available preparations, Endo-H may be derived from E. colitransformed with a plasmid encoding the Endo-H gene from Streptomycesplicatus and the promoter from alkaline phosphatase (Oka, T., et al.(1985) Proc. Natl. Acad. Sci. USA, 82, 7212-7216) by methods similar tothat reported for the cloning and expression of Endo-H from Streptomycesplicatus in E. coli (Robbins, et al. (1981) J. Biol. Chem. 256; 10 640).See also Trumbly R. J. et al. (1985) J Biol. Chem., 260, 5638. Endo-Hmay also be derived from Streptomyces cells engineered to express Endo-Hderived from Streptomyces plicatus (EPO Publication No. 0179449, Apr.30, 1986). Alternatively, Endo-H may be produced by any appropriate hostcell such as Bacillus subtilis using techniques well known to thoseskilled in the art. The amino acid and DNA sequences of Endo-H for S.plicatus (S. griseus) have been published. Robbins, P. W., et al. (1984)J. Biol. Chem., 159, 7577-7583.

The Endo-H used in the examples herein was obtained commercially or fromE. coli or B. subtilis hosts transformed to express Endo-H from S.plicatus.

One unit of Endo-H activity is the amount of enzyme required to release1 μmole of (³ H)-dansyl-Asn-GlcNAc from (³ H)-dansyl-Asn-(GlcNAc)₄(Man)₆ at pH 5.5 at 37° C. in one minute. Tarentino, A. et al. (1978)Methods in Enzymology, 50,574. The unit activity of other Type IIendoglycosidases are similarly defined by an appropriate substrate.

Of course, other Type II endoglycosidases may exist which have not yetbeen identified. Such Type II endoglycosidases as well as the onesdescribed herein, including allelic variations and geneticallyengineered modifications of such endoglycosidases are within the scopeof the present invention.

Glycosides and glycoside-containing substances often become bound to awide variety of surfaces. Thus, for example, glycoproteins, such asthose associated with blood (e.g., glycosylated hemoglobin), can stainthe surfaces of fabrics used for clothes, linen and the like. Suchstains have heretofore been highly resistant to complete removal bytreatment with detergents or detergents in combination with variousenzymes not comprising the endoglycosidases utilized in the presentinvention. A further glycoside-containing substance which stainssurfaces such as fabric and which is also difficult to remove by knowntechniques comprises fecal matter. Such fecal stains include variousglycosides and glycoside-containing substances associated withintestinal bacteria (e.g., peptidoglycans), catabolic excretions,including glycoproteins, and non-absorbed nutrients and the like.

Other surfaces to which glycosides or glycoside-containing substancesmay be bound include the surfaces of hard and soft contact lenses. Softcontact lenses are typically hydrophilic cross-linked polymers having ahydrogel structure or are made of silicon polymers. See, e.g., U.S. Pat.Nos. 3,403,393 and 2,976,576. Hard contact lenses, on the other hand,are typically made of methacrylate or methylmethacrylate polymers. Othersurfaces include naturally occurring biofilms, cardiac pacemaker leadsand power packs, cellular and mucosal surfaces, tooth enamel, filtersused to remove bacteria and particulate material in processing foods;chemicals and the like; air conditioning filters; the surfaces ofvarious structural components exposed to an aqueous environment, e.g.,boats, piers and the like; plastics and composites such as formica; andmetals or metal alloys such as steel, aluminum, etc.

As will be shown in detail hereinafter, Type II endoglycosidases aloneor in combination with a second enzyme such as subtilisin, either withor without detergent, effectively increases the removal of blood andfecal stains from cloth swatches. It is not known precisely how suchstains adhere to such swatches. However, the enhanced removal of suchsubstances from these swatches by Type II endoglycosidase, alone or incombination with other agents, suggests that at least one glycosidiclinkage is interposed between the fabric and that part of the stainwhich is released upon treatment with Type II endoglycosidase. Based onthese results, the following are proposed mechanisms of the binding ofglycoside-containing substances to a surface and the release and/orremoval of such substances by Type II endoglycosidase. These proposedmechanisms, however, should not be considered as a limitation to thescope of the invention.

FIGURES

Thus, as shown in FIG. 5A, a glycoside-containing substance may be boundto a surface other than by an immunological bond. In this regard, an"immunological bond" is one which exists between an antigen and anantibody, specific for that antigen (polyclonal or monoclonal). As shownin FIG. 5A, the glycoside-containing substance has a proximal portionbound to the surface and a distal portion extending outwardly from theproximal portion. The proximal and distal portions are joined by aglycosidic linkage with which Type II endoglycosidase is reactive. Asfurther shown in FIG. 5A, when treated with Type II endoglycosidase, thedistal portion of the glycoside-containing substance is "released" fromthe proximal portion of the glycoside-containing substance. To theextent that this distal portion is not bound by other means to thesurface, it is also readily "removed" from the surface and may be washedaway with a fluid.

In FIG. 5B, a glycoside-containing substance, in this case aglycoprotein containing a carbohydrate unit and protein unit, is shownbound to a surface. This glycoside-containing substance further containsa carbohydrate portion and an aglycon portion joined by a glycosidiclinkage which is reactive with Type II endoglycosidase. In thisparticular case, the glycoside-containing substance (glycoprotein) isbound to the surface through the carbohydrate portion of theglycoside-containing substance. When treated with Type IIendoglycosidase, the aglycon portion is released from the carbohydrateportion of the glycoside-containing substance. As in FIG. 5A, to theextent that the aglycon portion is not further bound to the surface byother means, the aglycon portion is also removed from the surface.

FIG. 5C depicts the situation where a glycoside-containing substance isbound to a surface by way of at least two points of attachment. Asindicated, a glycosidic first linkage exists between the surface and theglycoside-containing substance. In addition, a second linkage reactivewith a second enzyme is also present between the surface and the portionof the glycoside-containing substance to be removed. If treated onlywith Type II endoglycosidase, the portion of the glycoside containingsubstance distal from the first glycosidic linkage is released from thesurface at least to the extent that it was bound through the firstglycosidic linkage. If contacted with a second enzyme reactive with thesecond linkage shown, the portion of the glycoside-containing substanceas distal from the first glycosidic linkage and the second linkage isreleased from the surface. To the extent that this distal portion is nototherwise bound to the surface, i.e., by other contact points which maybe reactive with other enzymes or susceptible to detergents and/orsurfactants, this distal portion is effectively removed from thesurface.

FIG. 5D shows a microorganism bound to a surface through at least partof the glycoside portion of said microorganism. The glycoside portioncontains a glycosidic linkage reactive with Type II endoglycosidase. Acleaved portion of the microorganism distal from the glycosidic linkageis released from the surface when treated with Type II endoglycosidase.To the extent that this cleaved portion is not otherwise bound to thesurface it is also removed from the surface. However, multiple points ofcontact may exist with the surface which may require further treatmentwith other enzymes and/or detergent or surfactant.

In FIG. 5E, a Type II endoglycosidase-reactive substance is shown boundto a surface. This Type II reactive substance has a proximal portionbound to the surface and a distal portion extending outwardly from theproximal portion. The proximal and distal portions are joined by a TypeII reactive linkage which refers to a linkage reactive with a Type IIendoglycosidase. When treated with Type II endoglycosidase, the distalportion of the Type II reactive substance is "released" from theproximal portion of the Type II reactive substance. It is to beunderstood that Type II reactive substances may comprise molecules,microorganisms or aggregates of various components which may becomeattached to a surface. To the extent that the distal portion of the TypeII reactive substance is not bound by other means to the surface, it isalso readily "removed" from the surface and may be washed away with afluid.

The amount of Type II endoglycosidase used to produce the removal of thesubstances identified in the figures is defined functionally as an"amount effective" for removal of the particular substance from asurface. This amount may vary depending on the substance and surface tobe treated. Typical amounts are disclosed in more detail herein withregard to the specific embodiments disclosed.

SECOND ENZYMES

"Second enzymes" include proteases, lipases, glycosidases such aslysozyme and combinations thereof. Various proteases which may becombined with Type II endoglycosidase include subtilisin, bromilain,papaine, trypsin, chymotrypsin, pancreatin, lysozyme and combinationsthereof. Such enzymes may be derived from natural sources, e.g.,subtilisin from Bacillius subtilis or from genetically engineeredclones, e.g., subtilisin and mutant subtilisins as described in EPOPublication No. 0130756. See also, Wells, J. A., et al. (1983) NucleicAcids Res., 11, 7911-7915; Yang, M., et al. (1984) J. Bacteriology, 160,15-21; Estell, D. A., et al. (1985) J. Biological Chemistry, 260,6518-6521. Many such enzymes, of course, are available from commercialsources.

In addition, Type II endoglycosidases may be combined with lipases suchas bacterial, mammalian and fungal lipases and combinations thereof.

Glycosidases which may be used as a second enzyme includeexoglycosidases, a second Type II endoglycosidase and Type Iendoglycosidases. Examples include α- and β-amylase, cellulase,pectinase, hemicellulase, dextranase, various glucanases, and the likeand combinations thereof.

Moreover, Type II endoglycosidase may be combined with more than one ofthe above classes of second enzymes to facilitate the removal of aglycoside-containing substance from a surface.

When a Type II endoglycosidase is combined with one or more secondenzymes, the ratio of Type II endoglycosidase to second enzyme ispreferably about 0.01 to 100 and most preferably 1 to 1.

DISULFIDE CLEAVING REAGENTS

Type II endoglycosidases may also be used in combination withdetergents, either alone or in combination with one or more secondenzymes and/or disulfide-cleaving reagents to form a detergentformulation. Substances capable of cleaving disulfide bonds are varied,but fall generally into three categories: oxidizing agents, reducingagents, and miscellaneous addition substrates such as those exemplifiedby fumaric acid and sodium sulfite. Suitable oxidizing agents includehydrogen peroxide, performic acid, sodium perborate, and oxidizingbleaches. Effective reducing agents include dithiothreitol (DTT),β-mercaptoethanol (BME), sodium borohydride, and the like.

Alternate disulfide cleavage reagents which are not easily classifiedinclude mercuric chloride, nitroprusside, tributylphosphine, andphosphothiolate. A particularly useful cleavage reagent is sodiumsulfite, which results in sulfitolysis of the disulfide according to thereaction: R--S--S--R+SO₃ ⁻² R--S--SO₃ ⁻² +⁻ SR. The equilibrium of thisreaction may be shifted by removal of the thiol anion using heavy metalions or oxidizing agents. The oxidizing power may be provided byaeration or an oxidizing agent, such as CuSO₄ or sodium perborate.

The foregoing list of substances capable of cleaving disulfides is notmeant to be comprehensive, and conversely does include substances whichare effective but not necessarily appropriate for a commercial product.In order to be successful commercially, the added substance must berelatively inexpensive and must not have undesirable properties for itsintended use. Thus, for example, while the use of mercuric chloridewould be workable in carrying out the process of the invention, it wouldnot be suitable for ordinary detergent products intended for commercialuse. β-mercaptoethanol and DTT are feasible commercially, except thatthey have mildly offensive odors. Particularly preferred substances,therefore, for commercial formulation, are sodium sulfite (preferably incombination with an oxidizing agent) or hydrogen peroxide, which areinexpensive and are relatively safe. Reviews of materials which areuseful in the cleavage of disulfide bonds are found, for example, inChemical Modification of Proteins, Means, G. E., et al., eds (1971),Holden-Day, Inc. San Francisco, Calif., Ch 8; and Chemical Reagents forProtein Modification, Lundbald, R. L. et al., eds (1984), CRC Press,Inc., Boca Raton, Fla., Ch. 7.

Typically, the Type II endoglycosidase alone or in combination with oneor more second enzymes forms 0.01-3% wt/wt of the detergent compositionsof the invention, and may include disulfide-cleaving reagents, rangingfrom about 10-40% wt/wt thereof. The amounts present depend, of course,on the nature of the endoglycosidase (and second enzyme, if used) andthe disulfide cleavage reagent, the dilution of the detergent in thewash solution, and the conditions of the wash. However, the ranges givenare generally typical.

In one embodiment of the invention, surfaces having glycoproteincontaining substances bound thereto are treated with the combination(simultaneous or sequential) of a disulfide cleaving reagent, a Type IIendoglycosidase and a second enzyme at suitable pH, temperature, for anappropriate period of time. These conditions are, of course, variableaccording to convenience, and the selection of the Type IIendoglycosidase, protease and the substance to cleave disulfides to someextent depends on this selection. However, convenient conditionsfrequently encountered are pH values between 5 and 12. Temperatures of20°-55° C., particularly around 40°-55° C., and times of up to 20minutes, usually around 10-15 minutes are typical and preferred. Thepreferred times and temperatures are those generally utilized inhousehold washing machines, neighborhood laundromats, and professionallaundry services, since in order to be commercially practical, theprocess needs to be conducted under conditions ordinarily available tothe user.

In another embodiment of the invention, conventional washing proceduresusing commercial detergents are used and the Type II endoglycosidase,second enzyme and disulfide-cleaving substance are provided, eitherseparately or together, as an additive, much in the manner of themethods in which bleach is used. Thus, these may be added along with thedetergent at the beginning of the wash cycle or at some intermediatepoint, for example, after approximately half of the wash cycle iscompleted. If handled in this way, assuming an approximately 1:500dilution of a solid or liquid detergent composition (approximately 2mg/ml of the solid), arbitrary amounts of the Type II endoglycosidase,second enzyme and disulfide cleaving reagents may be added without theupper limit imposed by this dilution. (If the Type II endoglycosidase,second enzyme and disulfide cleaving reagent had been added to thedetergent composition originally, and if, for example, the disulfidecleaving reagent constituted 50% of the composition, only 1 mg/ml wouldresult in the final wash solution. However, if these materials are addedseparately, amounts most effective for the particular Type IIendoglycosidase, disulfide cleaving reagent and second enzyme may beadded.)

With respect to the Type II endoglycosidase and second enzyme, only verysmall quantities are usually required. Typically, the Type IIendoglycosidase and second enzyme are added to a final concentration ofapproximately 1-500 μg/ml of wash solution for each enzyme. In the caseof the disulfide-cleaving reagent, however, larger amounts than would bepermitted by the dilution of the detergent may be desirable. Forexample, cleavage of disulfide bonds using sodium borohydride mayconveniently be carried out with concentrations as high as 0.2M reagentin the present of similar quantities of buffer (Lundbald, R. L., et al.,Chemical Reagents for Protein Modification, supra).

Although such high amounts are conventional, they are not necessarilyrequired, and lower concentrations are workable. Sulfitolysis isordinarily carried out in sodium sulfite concentrations of the order of0.1M, although concentrations as low as 0.01M and lower can also beused. DTT is effective when supplied at concentrations of the order of0.02-0.1M. In short, the disulfide-cleaving reagent concentration can bevaried over a wide range for any of these reagents and effectivenessmaintained. The optimum concentration for a particular application will,of course, depend on the nature of the stain and the nature of thereagent, as well as the conditions of the wash procedure, includingtime, temperature, and pH.

In an alternative and more convenient approach, the Type IIendoglycosidase, second enzyme and disulfide-cleaving substance areadded to the original detergent composition, and the process isconducted as a standard wash procedure using these modified detergents.Under these circumstances, the detergent composition will correspond tothat described above, but the amount of the composition can also bevaried over the range of approximately 0.5 mg/ml-10 mg/ml or greater ofthe wash solution, depending, again, on the conditions of the washsolution and procedure, and on the solubilities of the detergentcomponents. In any case, the inclusion of the Type II endoglycosidase,disulfide-cleaving reagent and second enzyme in the detergent limits theconcentrations of these components in accordance with the dilution ofthe detergent. Thus, even if a 1:100 dilution is used (10 mg/ml), andthe disulfide-cleaving reagent for example, is limited to 50% of thedetergent composition, a maximum concentration of 5 mg/mldisulfide-cleaving reagent in the resulting wash solution is an upperlimit. Typically, of course, the concentration of disulfide-cleavingreagent in the detergent will be less than 50%, mandating even lowerconcentrations of the disulfide-cleaving reagent.

The detergent compositions of the invention contain mostly detergentactive substances, relatively smaller amounts of disulfide-cleavingreagent, if used, and quite small amounts of Type II endoglycosidase andsecond enzyme, if used, which is especially desirable in view of thecost of enzymic components. Thus, in general, the preparation willcontain 60-90% detergent active substances, including conventionalcommercial detergent additives such as surfactant builders andwhiteners, 0.01-3% Type II endoglycosidase and second enzyme, andapproximately 10-40% disulfide cleavage reagent.

Of course, it is also possible to add only one of these three additivesto the original detergent and to supply the other separately to the washliquid. In particular, the Type II endoglycosidase may be added to aprewash, followed by a detergent containing the second enzyme, oraddition of the detergent containing endoglycosidase may be followed orpreceded by treatment with the second enzyme.

CLEANING COMPOSITIONS

Endo D, F and H are preferred Type II endoglycosidases for use incleaning compositions. Endo-H is most preferred.

For removal of glycoside-containing substances, the compositions hereinpreferably comprise from about 0.1 ppm (parts per million) to 1200 ppm,more preferably from about 1 ppm to 1000 ppm, most preferably from about20 ppm to about 200 ppm, of Type II endoglycosidase, depending on thetype of composition. Cleaning compositions are preferred. Laundrydetergent compositions are most preferred for use herein, and preferablycomprise from about 0.1 ppm to 1200 ppm of Type II endoglycosidase,preferably from about 20 ppm to 200 ppm of Endo D, F or H, mostpreferably from about 50 ppm to 125 ppm Endo H.

When used to control or remove microorganisms, the compositionspreferably comprise from about 0.1 ppm to 1200 ppm, more preferably fromabout 1 ppm to 1000 ppm, most preferably from about 20 ppm to 400 ppm,of Type II endoglycosidase, preferably Endo-H. Cleaning compositions arepreferred and preferably comprise the same amounts of Type IIendoglycosidase, preferably Endo-H.

Described below are suggested types of compositions which comprise TypeII endoglycosidase for removal of glycoside-containing substances and/ormicroorganisms. The compositions can be made and used in any way whichdoes not destroy enzyme activity. They can be made up of any ingredientswhich do not unduly hinder the activity of the enzyme. The compositionscan be laundry detergents, dishwashing detergents, hard surfacecleaners, dental enamel cleaners, liquid and bar soaps, anti-acnecompositions, antiperspirants, shampoos, face creams, fruit andvegetable surface preservatives, or fabric softeners.

In addition to the cleaning of fabrics using common cycles in washingmachines, the cleaning compositions herein may also be used for removingglycoside-containing substances and/or microorganisms from othersurfaces such as metals and metal alloys such as found in surgicalinstruments, pipelines, metal containers and the like, and plastics andcomposite materials such as Formica and the surfaces of boats, piers andthe like. Depending upon the particular application, the composition maycomprise Type II endoglycosidase alone or in combination with adisulfide cleaving reagent, second enzyme and/or detergent surfactant.

Type II endoglycosidase may also be formulated in a composition forremoving glycoside-containing substances and/or microorganisms includingyeast, fungi, algae and bacteria from "biological surfaces" such assurfaces of skin, skin pores, hair, hair follicles and tissue. Thus,those skilled in the art of shampoo formulations, conditionerformulations, soap formulations and the medicinal arts can readily adaptthe above disclosure for detergent formulations to employ Type IIendoglycosidase in such applications. When so formulated, suchcompositions are useful in removing glycoside-containing substanceswhich may adhere to such surfaces.

Type II endoglycosidase may also be formulated in a composition forremoving glycoside-containing substances and/or microorganisms,especially yeast and fungus, from the surfaces of plants such as fruitsand vegetables. Such compositions preferably include nonionicsurfactant.

In addition, Type II endoglycosidase may be formulated in deodorantcompositions in a manner known to those skilled in the art to provideendoglycosidase activity to remove glycoside-containing substancesand/or microorganisms responsible for undesirable odors. Such deodorantformulations employing Type II endoglycosidase may include modificationsof formulations for stick, creams and aerosol deodorants known to thoseskilled in the art.

Further, Type II endoglycosidase may be formulated for the treatment ofacne which usually results from inflammation, at least to the extentthat glycoside-containing substances and/or microorgansims responsiblefor or involved in such inflamation are bound to a surface. As with theabove formulations, those skilled in the art are capable of modifyingknown acne formulations to incorporate a Type II endoglycosidase aloneor in combination with other enzymes, detergents and/or surfactants.

When used to treat contact lens, Type II endoglycosidase suitably issupplied at a concentration of about 0.1-20 μg/ml in the cleaningcompositions, and the concentration of a second enzyme such as aprotease is in the same range if such second enzymes are utilized.Treatment times can vary from about five minutes to about 15 hours, buta standard convenient cleaning time is overnight, so that the wearer canallow the lenses to soak while he sleeps. A variety of protocols aresuitable, but ones that are particularly preferred are the use of asingle solution containing Type II endoglycosidase and the second enzyme(if used) conducted from 10 minutes to two hours or overnight at roomtemperature, or a 10-minute to two-hour presoak in the presence of TypeII endoglycosidase solution, followed by a similar overnight treatmentwith a solution containing a second enzyme.

Preferred general purpose second enzymes for contact lens formulationinclude proteases such as papain, pancreatin and subtilisin. Thepreferred Type II endoglycosidase enzyme is Endo-H from Streptomycesplicatus. A single second enzyme protease may be used, or thecomposition may contain a mixture of second enzymes.

In addition, the contact lens compositions may include additionalcomponents which aid in the overall enzymatic degradation. Particularlyuseful among these are disulfide cleavage reagents such as2-mercaptoethanol, cysteine hydrochloride, dithiothreitol,dithioerythritol, sodium bisulfate, sodium metabisulfite, thiourea, andthe like, generally preferred in a range of about 0.01-5% by weightpreferably 0.05-1% by weight. In addition, detergents may be included inthe composition to aid in the wetting of the lens with theenzyme-containing solution. Suitable detergents include sodium dodecylsulfate, sodium monolaurate, nonionic surfactants such as alcoholethoxylates (e.g., polyethoxyethanol) anionic surfactants such as ethersulfonates, linear alkylbenzene sulfonates, sodium lauryl sulfate, andthe like.

Suitable buffers and stabilizers for contact lens cleaning may also beused and include sodium or potassium citrate, citric acid, boric acid,sodium EDTA, various mixed phosphate buffers and NaHCO₃. Generallybuffers and stabilizers may be used in amounts ranging from about 0.001to about 2.5% and preferably about 0.1 to 1% by weight. It should beunderstood that the foregoing description of the amounts of the variouscompounds which may be used in the present invention for cleaningcontact lens are stated in percentage of ingredients in solution(wt/vol). The formulation may also take the form of one or moreconventional solid dosage forms such as tablets suitable for use inmeasured quantity of a suitable solvent such as water. The percentagecomposition of the solid dosage forms is such that when dissolved in aspecified volume of water, the solution will have the percentagecomposition within the ranges set forth in the specification. If soliddosage forms are used, the formulation may include conventionallubricants, binders, and excipients which include glycerol, sorbitol,boric acid, propylene glycol, polyethylene glycols, dextran,methylcellulose, hydroxyethylcellulose, water soluble salts ofcarboxymethylcellulose, or naturally occurring hydrophilics such asgelatin, alginates, tragacanth, pectin, acacia and soluble starches.

Typical compositions and protocols useful in cleaning contact lensinclude the following:

1. The composition contains 1-100 μg/ml Type II endoglycosidase. Thelenses are removed and placed in contact with the solution for a periodof 12 hours at 22° C. The lenses are removed from the cleaning solutionand rinsed.

2. Solution A contains 10 μg/ml of Type II endoglycosidase; solution Bcontains 5 μg/ml subtilisin. The lenses are soaked in solution A for 30minutes at 25° C., removed, and immersed in solution B for 10 hours at25° C.

3. The cleaning solution contains 10 μg/ml of the protease pepsin and 10μg/ml of Type II endoglycosidase. The lenses are soaked in this solutionfor 5 hours at 20° C.

4. The cleaning solution contains 5 μg/ml subtilisin, 5 μg/ml Type IIendoglycosidase, and 10 mM 2-mercaptoethanol. The lenses are immersed inthis solution for 5 hours at 30° C.

5. The cleaning solution contains 7 μg/ml subtilisin, 3 μg/ml Type IIendoglycosidase, 10 mM 2-mercaptoethanol, and 2% sodium dodecyl sulfate(SDS). The lenses are soaked in this solution for 3 hours at 20° C.

6. The cleaning solution contains 4 μg/ml subtilisin, 2 μg/ml trypsin,10 μg/ml Type II endoglycosidase, and 2% SDS. The lenses are soaked inthis solution for 7 hours at 20° C.

7. Solution A contains 4 μg/ml subtilisin and 2 μg/ml trypsin in 2% SDS.Solution B contains 10 μg/ml Type II endoglycosidase plus 10 mM2-mercaptoethanol. The lenses are immersed in solution B for 20 minutesat 30° C. and then in solution A for 6 hours at 25° C.

In all the foregoing examples, the lenses are thoroughly rinsed insaline before being returned to the wearer's eyes.

The compositions herein can be formulated in a variety of physicalforms, including liquids, gels, pastes and solid particles such aspowders and granules. The compositions can be formulated as laundrydetergents, such as disclosed in U.S. Pat. Nos. 4,507,219, 4,318,818,4,605,509 and 4,412,934; dishwashing detergents such as disclosed inU.S. Pat. Nos. 4,714,562, 3,630,923, 4,133,779, 4,316,824 and 4,555,360;hard surfaces cleaners such as disclosed in U.S. Pat. Nos. 4,414,128,3,679,608, 3,985,668 and 4,005,027; fabric softeners such as disclosedin U.S. Pat. Nos. 3,944,694, 4,073,996, 4,424,134 and 4,661,269; barsoaps such as disclosed in U.S. Pat. Nos. 3,993,722 and 3,070,547;shampoos such as disclosed in U.S. Pat. Nos. 4,345,080, 4,704,272 and4,741,855; antiperspirants such as disclosed in U.S. Pat. No. 4,725,432;anti-acne products such as disclosed in U.S. Pat. Nos. 4,318,907 and4,608,370; and oral compositions such as disclosed in U.S. Pat. No.4,684,518. The above patents are incorporated herein by reference.

The compositions preferably have a pH from about 4 to 10, morepreferably from about 5 to 8 for good enzyme performance.

Laboratory work on microorganism removal has shown that, in order toobtain effective removal, the bathing of the surface holding themicroorganisms in some instances requires a physical or chemical actionto remove the microorganisms. Microorganisms tested include:

Escherichia coli including Type 1 and 3 fimbriae

Staphylococcus aureus

Staphylococcus epidermidis

Serratia marcescens

Streptococcus mutans

Streptococcus sanguis

Bacillus sp.

Candida sp.

Aspergillus sp.

In the case of removal of bacteria such as E. coli, for example, thesurface-bound microorganisms may be treated with Endo-H and then removedby chemical action, such as by treatment with an antimicrobial agent, ora physical action, such as by rinsing with water or hand wiping. It ispreferred for liquid and bar soaps, dental enamel cleaners,antiperspirants, anti-odor fabric softeners and anti-acne compositionsthat the composition include an anti-microbial agent, such as Irgasan®(Ciba-Geigy) or chlorhexidine, in addition to the Endo-H. Anantimicrobial agent is not required in the composition (for example ahard surface cleaner) when physical action such as water rinsing orwiping by hand will occur.

Preferred herein are detergent cleaning compositions, especiallygranular and liquid laundry detergent compositions. These detergentcleaning compositions preferably comprise from about 1% to 90%, morepreferably from about 5% to 50%, by weight, of detergent surfactants,most preferably from about 10% to 40% by weight.

Surfactants useful in the detergent compositions herein includewell-known synthetic anionic, nonionic, amphoteric and zwitterionicsurfactants. Typical of these are the alkyl benzene sulfonates, alkyl-and alkylether sulfates, paraffin sulfonates, olefin sulfonates,alkoxylated (especially ethoxylated) alcohols and alkyl phenols, amineoxides, alpha-sulfonates of fatty acids and of fatty acid esters, alkylbetaines, and the like, which are well known from the detergency art. Ingeneral, such detersive surfactants contain an alkyl group in the C₉-C₁₈ range. The anionic detersive surfactants can be used in the form oftheir sodium, potassium or triethanolammonium salts; and the nonionicsurfactants generally contain from about 5 to about 17 ethylene oxidegroups. C₁₁ -C₁₆ alkyl benzene sulfonates, C₁₂ -C₁₈ paraffin-sulfonatesand alkyl sulfates are especially preferred in the compositions of thepresent type.

A detailed listing of suitable surfactants for the compositions hereincan be found in U.S. Pat. No. 3,936,537, Baskerville, issued Feb. 3,1976, incorporated by reference herein. Commercial sources of suchsurfactants can be found in McCutcheon's Emulsifiers and Detergents,North American Edition, 1984, McCutcheon Division, MC PublishingCompany, also incorporated herein by reference.

Useful detergency builders for the detergent compositions herein includeany of the conventional inorganic and organic water-soluble buildersalts, as well as various water-insoluble and so-called "seeded"builders. The instant laundry detergent compositions preferably comprisefrom about 1% to 75%, more preferably from about 5% to 40%, mostpreferably from about 10% to 20%, by weight of detergent builders. Thesecompositions preferably have a pH of from about 6 to 10.

Nonlimiting examples of suitable water-soluble, inorganic alkalinedetergent builder salts include the alkali metal carbonates, borates,phosphates, polyphosphates, tripolyphosphates, bicarbonates, silicatesand sulfates. Specific examples of such salts include the sodium andpotassium tetraborates, bicarbonates, carbonates, tripolyphosphates,pyrophosphates, and hexametaphosphates.

Examples of suitable organic alkaline detergency builder salts are: (1)water-soluble amino polyacetates, e.g., sodium and potassiumethylenediaminetetraacetates, nitrilotriacetates, andN-(2-hydroxyethyl)nitrilodiacetates; (2) water-soluble salts of phyticacid, e.g., sodium and potassium phytates; (3) water-solublepolyphosphonates, including sodium, potassium and lithium salts ofethane-1-hydroxy-1,1-diphosphonic acid, sodium, potassium, and lithiumsalts of methylenediphosphonic acid and the like.

Seeded builders include such materials as sodium carbonate or sodiumsilicate, seeded with calcium carbonate or barium sulfate. Hydratedsodium zeolite A having a particle size less than about 5 microns isparticularly desirable.

A detailed listing of suitable detergency builders can be found in U.S.Pat. No. 3,936,537, incorporated herein by reference. Preferred buildersare fatty acids, polycarboxylates, polyphosphates and mixtures thereof.

Optional detergent composition components include enzymes (e.g.,proteases and amylases), peroxygen bleaches and bleach activators,halogen bleaches (e.g., sodium and potassium dichloroisocyanurates),soil release agents (e.g., methylcellulose), soil suspending agents(e.g., sodium carboxymethylcellulose), fabric brighteners, enzymestabilizing agents, color speckles, suds boosters or suds suppressors,anticorrosion agents, dyes, fillers, germicides, pH adjusting agents,nonbuilder alkalinity sources, and the like.

ENDOGLYCOSIDASE PLUS ANTIMICROBIAL AGENTS

Of the Type II endoglycosidases, endo-β-N-acetylglucosaminidase H, D, Fand/or PNGase F are preferred for formulating antimicrobial compositionsand for use in the antimicrobial methods herein. Endo-H is mostpreferred.

When the Type II endoglycosidase is used alone, it is formulated suchthat its concentration produces an antimicrobial effect. When theantimicrobial composition comprises at least two different components,i.e. a Type II endoglycosidase and one or more antimicrobial agents,each of the components are present at a concentration sufficient toproduce an antimicrobial effect. The amount of at least one component insaid compositions is generally less than the amount required for thatcomponent to produce the same antimicrobial effect if used alone in asimilar composition.

As used herein, an "antimicrobial effect" includes the removal, killing,inhibition of growth, change in gross morphology, protoplast formationand/or degradation of the cell wall of a microorganism when contactedwith a Type II endoglycosidase alone or in combination with a secondcomponent comprising an antimicrobial agent.

As used herein, an "antimicrobial method" refers to a method whichproduced an antimicrobial effect. In one aspect of the invention, theantimicrobial method causes the killing of microorganisms, theinhibition of microorganism growth, and/or changes in the grossmorphology of the microorganism. In another aspect of the invention, theantimicrobial method causes the removal of a microorganism from asurface. In the antimicrobial methods to remove microorganisms fromsurfaces, it is preferred that the surface be treated with theantimicrobial agent and the Type II endoglycosidase simultaneously,rather than treating with the additional antimicrobial agent immediatelyafter treating with Type II endoglycosidase. In some applications of theantimicrobial methods, a combined antimicrobial effect may be produced,e.g. killing and/or growth inhibition may occur in combination withmicroorganism removal from a surface.

As used herein, an "antimicrobial composition" refers to a compositioncontaining at least two different components: a Type II endoglycosidaseand a different component comprising an antimicrobial agent. Suchantimicrobial compositions have variable antimicrobial effects dependingupon the amount and choice of Type II endoglycosidase and antimicrobialagent. Observed antimicrobial effects include the killing ofmicroorganisms and/or inhibiting microorganism growth, the removal ofmicroorganisms from a surface and the prevention of microorganismattachment to surfaces.

As used herein, an "antimicrobial-effective concentration" of Type IIendoglycosidase generally refers to the final concentration of Type IIendoglycosidase used alone to contact a microorganism to produce anantimicrobial effect.

As used herein, an "antimicrobial agent" is a second different componentof an antimicrobial composition. Such antimicrobial agents in generalare antibiotics and include agents which kill microorganisms and thosewhich inhibit microorganism growth. Examples of such antimicrobialagents include bacteriocides, fungicides and algicides each of which arecapable of killing or inhibiting the growth of bacteria, fungi or algae,respectively. Bacteriocides include compounds such as chlorhexidine,2,4,4'-trichloro-2'-hydroxydiphenyl ether, Triclocarban®, penicillins,tetracycline and Bacitracin®. Fungicides include Nystatin®, AmphotericinB®, Benomyl®, Captan® and Dichloran®. Other examples of antimicrobialagents include surfactant-stable antimicrobial enzymes such assurfactant-stable β-1,3-glucanases, lysozymes, proteases and chitinases,and detergent surfactants such as anionic, nonionic, zwitterionic,ampholytic and cationic surfactants known to those skilled in the art.The latter should be employed in an amount sufficient to produce anantimicrobial effect. The above antimicrobial agents identified bygeneric name or trademark are compositions as identified in the MerckIndex, 10th Ed. (1983), Merck & Co., Inc., Rahway, N.J.

Type II endoglycosidases different from the first component of theantimicrobial compositions may also be used as an antimicrobial agent.Thus, to the extent Type II endoglycosidases are themselvesantimicrobial agents (e.g. are capable of producing an antimicrobialeffect, such as morphological changes or protoplast formation), they maybe combined with a different Type II endoglycosidase to form anantimicrobial composition. Antimicrobial compositions may thereforecomprise one or more different Type II endoglycosidase with or withoutone or more antimicrobial agents not comprising Type II endoglycosidase.

Preferred antimicrobial agents for use herein are chlorhexidine,2,4,4'-trichloro-2'-hydroxydiphenyl ether, Triclocarban®, Nystatin®Amphotericin B® antibiotic, anionic and nonionic detergent surfactants.A surfactant-stable antimicrobial lysozyme is disclosed in U.S. Pat. No.5,041,236 entitled Methods and Compositions Employing Certain Lysozymesand Endoglycosidases in the names of Richard S. Carpenter and Ann M.Wolff, filed on even date as this application. Other lysozymes, e.g. henegg white lysozyme, have been used in combination with Endo-H to produceantimicrobial effects albeit to a lesser extent and with variability inthe results obtained.

The antimicrobial compositions and methods of the invention can producean antimicrobial effect on a wide range of microorganisms includingGram-positive and negative bacteria, fungi, and algae. Such bacteriainclude Escherichia coli, Streptococcus mutans, Staphylococcusepidermidis, and Staphylococcus aureus. Such fungi include yeasts suchas Candida and Saccharomyces, and species and filamentous fungi such asAspergillus, Sporobolomyces, Basidiobolus and Entomophthora.

A specific advantage of combining a Type II endoglycosidase (e.g.Endo-H, D, F and/or PNGase F) with an antimicrobial agent is that lessof the antimicrobial agent can be used to produce an antimicrobialeffect. In some aspects of the invention, the antimicrobial agent whenused with a Type II endoglycosidase produces an antimicrobial effectcomprising the removal of microorganisms attached to surfaces or theprevention of their attachment to such surfaces. In other aspects, thereis a negative effect on microorganism viability or microorganismmorphology.

Surface treatment(s) with Type II endoglycosidase and antimicrobialagent can be performed periodically so as to prevent further growth orattachment or adhesion of microorganisms to the surfaces exposed to thetreatment.

Of the Type II endoglycosidases, Endo-H, D, F and/or PNGase F arepreferred. Of these, Endo-H is most preferred. In general, anantimicrobial-effective amount of Type II endoglycosidases for use incombination with antimicrobial agents is from about 1 to 1200 ppmEndo-H, D, F, and/or PNGase F, preferably from about 1 to 1200 ppmEndo-H, more preferably from about 20 to 1000 ppm Endo-H, mostpreferably from about 50 to 400 ppm Endo-H. The amount used depends uponthe type of treatment and amount of exposure to the surface ormicroorganism to be treated. In general, an effective amount ofantimicrobial agent, which depends upon which agent is used, is fromabout 0.5 to 1200 ppm, preferably 2 to 1200 ppm, most preferably fromabout 5 to 350 ppm chlorhexidine or 2,4,4'-trichloro-2'-hydroxydiphenylether, or 0.5 to 100 ppm Nystatin®.

When Type II endoglycosidase is used alone to kill and/or inhibitmicroorganisms, the use of substantially more Type II endoglycosidase isgenerally required. For example, about 100 ppm to 1000 ppm of Endo-H hasbeen shown to substantially decrease the viability of yeast cellsexposed to such concentrations. When yeast is exposed to less than 100ppm of Endo-H, however, a significant decrease in viability has not beenobserved. Although the lower limit of Endo-H necessary to adverselyaffect yeast viability has not yet been determined, the lower limit ofits antimicrobial-effective concentration is believed to be between 10and 100 ppm. Similar amounts of Endo-H are believed to be useful to killand/or inhibit other microorganisms such as algae and fungi. The exacteffect of Endo-H and other Type II endoglycosidases on these organismsand others, e.g., bacteria, when not used in combination withantimicrobial agents has not yet been determined. The range ofantimicrobial-effective concentrations of Type II endoglycosidase foruse against such organisms, however, can be routinely determined.

The antimicrobial methods and compositions of the invention have a wideapplicability and include antimicrobial methods and compositions forpersonal care, health care and household and industrial cleaning. Thus,such methods and compositions may be used to formulate and useantimicrobial mouthwash, dentifrice or denture cleaner, as well asantimicrobial liquid or solid hand or body soaps, anti-acne medication,deodorant, shampoo and face creams and compositions for cleansing woundsor suppressing infections. Typical household applications includeantimicrobial cleaning products such as liquid soap, hard surfacecleaners, and liquid and granular laundry detergents. Heavy dutyantimicrobial detergent compositions may also be formulated forindustrial use.

Chlorhexidine is an effective oral antibacterial agent and is preferredfor use in dental applications. 2,4,4'-trichloro-2'-hydroxydiphenylether is available as Irgasan® DP 300 from Ciba-Geigy and is abroad-spectrum antimicrobial effective in personal care and laundryapplications. Triclocarban® from Monsanto is a bacteriostat useful inbar soaps. Traditional antibiotics can also be employed as theadditional antimicrobial agent herein. Lastly, surfactant-stableantimicrobial enzymes can be used in dental applications and forpreservation of shampoos and other surfactant-containing formulations. Apreferred surfactant-stable antimicrobial enzyme is the lysozymedisclosed in the previously identified copending application in thenames of Carpenter and Wolff. Surfactant-stability of antimicrobialenzymes can be gauged herein by retained activity in the presence ofrepresentative amounts of alkyl ether sulfate or linear alkylbenzenesulfate, for example.

The antimicrobial composition may be formulated as an antimicrobialmouthwash, dentifrice, or denture cleaner. The treatment ofmicroorganisms to produce an antimicrobial effect (e.g. to remove orprevent microorganism attachment to natural or synthetic soft and/orhard surfaces in the oral cavity or to kill microorganisms or inhibittheir growth in the oral cavity), then, essentially comprises rinsingwith an antimicrobial mouthwash, cleaning the teeth with anantimicrobial dentifrice, and/or cleaning dentures with an antimicrobialdenture cleaner. The antimicrobial mouthwash, dentifrice and denturecleaners herein preferably comprise Endo-H, and chlorhexidine and/orsurfactant stable antimicrobial enzyme as the antimicrobial agent. Wherechlorhexidine is used, the antimicrobial mouthwash, dentifrice, ordenture cleaner preferably comprises from about 50 to 1200 ppm Endo-Hand from about 50 to 350 ppm chlorhexidine. Where surfactant-stableantimicrobial enzyme is used, the antimicrobial mouthwash, dentifrice ordenture cleaner preferably comprises from about 50 to 150 ppm Endo-H andfrom about 50 to 1,000 ppm surfactant-stable antimicrobial enzyme.

The antimicrobial composition may also be formulated as antimicrobialpersonal care or household cleaning products. In such products, Endo-His preferably used at a concentration of from about 1 to 1200 ppm. Theantimicrobial agent for use in these products is preferablychlorhexidine, most preferably at a concentration of from about 150 to1200 ppm, or 2,4,4'-trichloro-2'-hydroxydiphenyl ether, most preferablyat a concentration of from about 2 to 500 ppm. Preferred personal careor household cleaning products are liquid hand soaps, hard surfacecleaners, laundry detergents and shampoo (described below).

A preferred antimicrobial liquid hand soap comprises from about 50 to400 ppm Endo-H, from about 5 to 100 ppm2,4,4'-trichloro-2'-hydroxydiphenyl ether, and preferably from about 1to 40 weight % to detergent surfactant. Preferably from about 2 to 20weight %, most preferably from about 3 to 10 weight %, detergentsurfactant is employed, preferably selected from the group consisting ofanionic, nonionic, zwitterionic, ampholytic and cationic surfactants.The liquid hand soap can further comprise emollient (up to about 30weight %) and minor amounts of perfume, colorant, solvent, andopacifier.

The antimicrobial hard surface cleaners herein can be glass cleaners,abrasive hard surface cleaners, scouring cleansers, or toilet bowelcleaners. These should be substantially free of hypochlorite-generatingbleaches, and other endoglycosidase-incompatible ingredients. Apreferred hard surface cleaner comprises from about 100 to 1000 ppmEndo-H, and antimicrobial agent, and from about 0.1 to 20 weight %detergent surfactant. From about 2 to 10 weight %, detergent surfactantis most preferred, preferably selected from the group consisting ofanionic, nonionic, zwitterionic, ampholytic and cationic surfactants.The antimicrobial hard surface cleaners herein optionally furthercomprise abrasive, builder, diluent, solvent, suspending agent (such asclay, carboxymethylcellulose, and polyacrylate), perfume, and/orcolorant.

The antimicrobial laundry detergent herein, in addition to Type IIendoglycosidase and antimicrobial agent, preferably comprises from about1 to 99 weight %, more preferably from about 5 to 60 weight %, mostpreferably from about 10 to 40 weight % detergent surfactant, preferablyselected from the group consisting of anionic, nonionic, zwitterionic,ampholytic and cationic surfactants. A preferred liquid or granularantimicrobial laundry detergent comprises from about 2 to 250 ppmEndo-H, from about 2.5 to 40 ppm 2,4,4'-trichloro-2'-hydroxydiphenylether, and from about 1 to 99 weight %, preferably from about 5 to 60weight %, detergent surfactant. The antimicrobial laundry detergentsherein optionally further comprise builder, perfume, bleach, diluent,suds suppressor, colorant, brightener, soil suspending agent,antiredeposition aids, softeners, and/or soil release agents.

The antimicrobial shampoo for use herein preferably comprises Endo-H, anantimicrobial agent, and from about 5 to 60 weight % detergentsurfactant, preferably selected from the group consisting of laurylsulfate, isoethionate, acyl amidobetaine, alkyl glyceryl ethersulfonate, and alkyl ether sulfate. Optional ingredients are sudsbooster, conditioner, dye, colorant, perfume and/or anti-dandruff agent.

The present antimicrobial compositions may also be in the form of apreservative or microorganism control agent for treatment of plantsurfaces. Preferred are a preservative for the surfaces of fruits orvegetables or an antimicrobial product to be applied on crops formicroorganism control. The latter is preferably in the form of asolution to be sprayed on crops such as corn, citrus, wheat, tobacco,soybeans, tomatoes and strawberries for control and prevention ofmicroorganism growth.

The following is presented by way of example only and is not to beconstrued as limiting the scope of the invention.

EXAMPLE 1 Removal of Blood and Fecal Matter from Fabric

Separate blood and fecal matter stained (cotton fabric) swatches werewashed with commercial detergents in an automatic washing machine usinga warm (approximately 37° C.) wash cycle. The swatches were then rinsedand air dried. They were then incubated with various amounts and typesof endoglycosidase [(0.005 U of Endo-D (Boehringer MannheimBiochemical), or Endo-H (Boehringer Mannheim Biochemical from S.griseus, Catalog No. 752 967), and 0.25U N-glycanase (PNGase F orpeptide endoglycosidase F) Genzyme, Boston, Mass.] in 0.75 ml of 50 mMTris-HCl, pH 7.0 at 37° C. for 30 minutes in a test tube. The controlcontained buffer but no endoglycosidase. At the end of the incubationperiod, 0.25 ml of detergent solution (1:125 dilution of a commercialliquid detergent composition which did not contain dyes, perfumes,enzymes or brighteners in 1M Tris-HCl, pH 7.5) containing 80 ug ofsubtilisin BPN'/ml was added to the control and enzyme containingsamples and incubated for an additional 20 minutes. At the end of thistreatment, the tubes were centrifuged and the protein content in thesupernatants were determined by measuring absorbance at 280 nm. For eachtreatment, a reaction blank was prepared which contained no swatchduring the assay. The blank values were subtracted from the absorbanceof treated samples to determine the release of 280 nm absorbing materialduring incubation. Higher absorbance represents increased release ofprotein from fibers. The results are shown in Table III.

                  TABLE III                                                       ______________________________________                                                   Absorbance at 280 nm                                               Treatment    Blood Stain                                                                             Fecal Matter Stain                                     ______________________________________                                        Control      0.79      2.07                                                   Endo-D       0.84      2.14                                                   Endo-H       0.83      2.12                                                   N-Glycanase  0.78      2.10                                                   ______________________________________                                    

These results suggested that the endoglycosidases, Endo-D and Endo-H, incombination with the second enzyme subtilisin increased the release of280 nm absorbing material from the blood stained swatches as compared tothe control. In addition, Endo-D, Endo-H and N-glycanase all showed anincrease in the release of 280 nm absorbing material from the fecalstained swatches.

EXAMPLE 2 Effect of Endo-H on Removal of Fecal Matter Stain

This example is similar to Example 1 but was performed by using fecalmatter stained swatches made of nylon fabric. The swatches were washedin detergent solution, rinsed and dried. The detergent consisted ofliquid commercial detergent which did not contain enzymes, brighteners,dyes or perfumes. One set of swatches was kept aside and referred as"untreated control". These swatches were treated the same as the sampleswatches except that they were not treated with Endo-H. The sampleswatches were incubated with 0.01U Endo-H (Boehringer MannheimBiochemical Catalog No. 752 967) in buffer (10 mM Na-acetate, pH 6.0) at37° C. for 15 minutes. Then 0.25 ml of detergent solution (1:125dilution in 1.0M Tris-HCl, pH 7.5) was added and incubated for anadditional 15 minutes. At the end, tubes were centrifuged and thesupernatants removed by suction. The swatches were air dried. Fibersfrom the swatches were examined by scanning electron microscopyfollowing critical point drying. An electron micrograph of adetergent-washed swatch stained with fecal matter is shown in FIG. 6A.As can be seen, rod like bacteria and particulate matter are found onthe surface of the fabric. FIG. 6B shows a swatch treated with Endo-Hand detergent. This figure shows a smooth clean fabric whichdemonstrates that Endo-H and detergents facilitates the removal ofparticulate material and bacterial debris.

EXAMPLE 3 Effect of Endo-F on Fecal Matter Stain

Swatches stained with fecal matter (1 inch diameter) were washed indetergent solution, rinsed and dried. Swatches were cut into quartersand used in the following experiments.

A. Swatches were incubated in 1 ml 10 mM sodium-acetate buffer, pH 5.5with or without Endo-F (Boehringer Mannheim Biochemical) (0.15 units)for 30 minutes at 37° C. The tubes were then centrifuged for eightminutes. Supernatants were removed and the absorbance of each wasmeasured at 280 mm. Change in A280 was determined by subtractingappropriate blanks (see Example 1). Higher absorbance includes theincrease in the amount of protein or material absorbing at 280 mmreleased from the swatches. For the controls, the average change in A280was 0.93. For swatches treated with Endo-F the average change in A280was 1.05. This indicated that Endo-F increases the efficiency of fecalstain removal.

B. Swatches were incubated in 0.75 10 mM sodium-acetate buffer pH 5.5with or without Endo-F (0.15 units) for 15 minutes at 37° C. At the endof this treatment, 0.25 ml of detergent solution (in 0.1M Tris-HCl, pH7.5) containing 10 μg of the protease subtilisin BPN' was added and thetubes were incubated at 37° C. for another 15 minutes. At the end, tubeswere centrifuged, supernatants were removed and absorbance at 280 nm wasmeasured. In the case of the control (no Endo-F), the average change inA280 was 1.08 whereas the sample treated with Endo-F showed a change inA280 of 1.36. This indicated that the effect of Endo-F was enhanced bythe presence of the detergent.

C. An experiment similar to "B" was performed except the detergentsolution contained 10 mM 2-mercaptoethanol instead of subtilisin. Theaverage change in A280 for the control was 1.05 whereas the sampletreated with Endo-F produced a change in A280 of 1.24. These resultsdemonstrated the ability of Endo-F in the presence of disulfide cleavingreagents to remove fecal stains.

D. An experiment similar to "B" was performed except that the detergentsolution contained 10 mM 2-mercaptoethanol and 10 μg subtilisin BPN'.The average change in A280 for the control was 1.14 whereas the Endo-Ftreated sample had a change in A280 of 1.29. These results indicate thatEndo-F is capable of removing fecal matter in the presence of detergent,a protease and a disulfide cleaving reagent (2-mercaptoethanol).

EXAMPLE 4 Comparison of Endo-H with Other Enzymes

Experiments similar to those described in part B of Example 3 wererepeated with Endo-H (Boehringer Mannheim Biochemical Catalog No. 100119) and other carbohydrase enzymes except that no protease such assubtilisin was used. Changes in A280 were monitored and fibers wereexamined by scanning electron microscopy. Removal of particulate andbacterial debris from fabric was seen with Endo-H and "Lysing Enzymes"(a mix of proteases and glyconases obtained from Sigma ChemicalCompany). However, the enzymes, lysozyme, α-glycosidase, β-glucosidaseand β-glucorinadase, showed little or no benefit. (Results not shown.)The results of electron microscopy for this experiment for treatmentwith or without the above enzymes are shown in FIGS. 7A through 7H. FIG.7A is a control which was not treated with endoglycosidase. FIG. 7B isan electron micrograph of a swatch treated with lysozyme; FIG. 7C is aswatch treated with Endo-H; FIG. 7D is a swatch treated withα-glucosidase; FIG. 7E is a swatch treated with β-glucosidase; FIG. 7Fis an electron micrograph of a fiber treated with "Lysing Enzymes"; FIG.7G is an electron micrograph of a swatch treated with β-glucorinadase;and FIG. 7H is an electron micrograph of a swatch treated withchitinase. As can be seen, the swatch treated with Endo-H (FIG. 7C) hasbeen thoroughly cleansed of the fecal matter stain. Similar results wereobtained for the swatches treated with "Lysing Enzymes" as shown in FIG.7F.

EXAMPLE 5 Removal of Bacteria from a Solid Surface

To test the effect of Endo-H on removal of bacteria from solid surfaces(glass),the following protocol was used. Trypticase soy broth (TSB) (10ml) was inoculated with a microbial species (Staphylococcus aureus ATCCculture #6538 or Escherichia coli ATCC culture #10536) from a stockculture slant and incubated overnight at 37° C. A suspension of about10⁸ cells/ml TSB was prepared and 100 μl of this suspension was placedwithin the etched ring on a glass slide. Each slide was incubated for 5minutes at 37° C. in a dry incubator oven after which excess microbialsolution was tapped off. The slides were then rinsed with 100 μl ofsterile distilled water. The excess solution and loose organisms werethen tapped off.

After the bacteria were adhered to the glass slides (2 or more hours at37° C.), 100 μl of the following solutions were applied to separateslides: (a) 10 mM acetate buffer, pH 5.5, (b) 10 mM acetate buffer, pH5.5+1 ppm Endo-H (Boehringer Mannheim Biochemical Catalog No. 100 119),(c) detergent solution, (d) detergent solution+1 ppm Endo-H. A set ofslides were kept aside as untreated controls and were not treated withany solutions. The non-control slides were then incubated for 15 minutesat 37° C. At the end of the incubation, the solutions were tapped off.The slides were then rinsed with 100 μl of sterile distilled water andair dried at room temperature. The bacteria which remained after thistreatment were heat fixed and stained by a standard Gram stainingmethod. The slides were then examined by a light microscope (brightfield illumination, 125× magnification) and the number oforganisms/field was determined. Twenty fields were counted for eachslide from which the average organisms/field was calculated.

The following results were obtained:

    ______________________________________                                        A. For Staphylococcus aureus                                                  ______________________________________                                        i)    No treatment   >100 organisms/field                                     ii)   Buffer         >100 organisms/field                                     iii)  Buffer + Endo-H                                                                               <10 organisms/field                                     iv)   Detergent solution                                                                           >100 organisms/field                                     v)    Detergent + Endo-H                                                                            <10 organisms/field                                     ______________________________________                                    

These results indicate that Endo-H buffer alone or in combination withdetergent reduced the number of S. aureus bacteria retained on the glassslides 10 fold as compared to treatment with detergent alone.

    ______________________________________                                        B. For Escherichia coli                                                       ______________________________________                                        i)    No treatment   >100 organisms/field                                     ii)   Buffer         >100 organisms/field                                     iii)  Buffer + Endo H                                                                              >100 organisms/field                                     iv)   Detergent      >100 organisms/field                                     v)    Detergent + Endo H                                                                            <10 organisms/field                                     ______________________________________                                    

These results indicate that Endo-H in combination with a detergentreduced the number of E. coli retained on the glass slide 10-fold ascompared to treatment with detergent alone.

EXAMPLE 6 Removal of Bacteria from a Solid Surface

An experiment similar to Example 5 was performed with twoslime-producing Staphylococcus aureus cultures (determined by theirabilities to bind to polystyrene tubes). Microscope slides were modifiedby forming two rings (≃1.7 cm diameter) with nail polish. Overnightculture of the organisms were diluted 1:10 with 1% peptone solution.Diluted culture (100 μl) was put in rings. Slides were put in 150 cmpetri dishes and incubated at 37° C. After two hours incubation, slideswere rinsed with distilled water and treated wth three differentconditions (A. Na-acetate buffer, B. detergent, and C. detergent plus 1μg Endo-H/ml) as in Example 5. The Endo-H was obtained from E. colitransformed to produce Endo-H from S. plicatus. At end of 15 minutes,incubation slides were rinsed with distilled water and Gram stained. Thenumber of bacteria was counted under microscope per 100× field for 20fields. The results are expressed as the average number of cells perfield.

    ______________________________________                                        Condition         Culture I                                                                              Culture II                                         ______________________________________                                        A.    Control         23       202                                            B.    Detergent       9        58                                             C.    Detergent + Endo-H                                                                            2        33                                             ______________________________________                                    

EXAMPLE 7 Removal of Bacteria from a Cloth Surface

To test the effect of Endo-H on the removal of bacteria from a clothsurface, the following protocol was used. Staphylococcus aureus (ATCC6538) and Staphylococcus epidermidis (ATCC 155) were separately culturedin 5 ml of Luria's broth and allowed to grow at 37° C. for 12 hours. Thecultures were then added to 30 ml of 0.2M NaCitrate, pH 5.5 buffer atabout 10³ cells/ml, in two 100 ml shake flasks. Twelve cloth swatches(0.5×0.5 inch cotton swatches) were also added to the flasks afterinoculation. After incubation at 37° C. for two hours with gentlerotation (150 rpm), the swatches were transferred to sterile tubes andwashed 3× with buffer comprising 200 mM NaCitrate, pH 5.5 which had beenpreviously sterilized by 0.22 micron filtration. Six swatches were thenadded to a shake flask containing 0.5 mg/ml Endo-H in 30 ml citratebuffer, and six swatches were added to a shake flask containing onlycitrate buffer as the control. The Endo-H was obtained from E. coliproducing S. plicatus Endo-H. After incubation at 37° C. for 1.5 hourswith gentle rotation (100 rpm), the swatches were transferred to steriletubes and washed as previously described. Swatches were then platedcarefully on trypticase soy agar plates and overlaid with enough liquidtrypticase soy agar to cover the swatches. After the plates were dry,they were incubated at 37° C. for 18 hours, and colonies ofStaphylococcus aureus and Staphylococcus epidermidis on the clothsurface were counted using a dissecting scope.

The following results were obtained:

    ______________________________________                                        A. For Staphylococcus aureus                                                  ______________________________________                                        Control   103 +/- 24 colonies per swatch                                      Endo-H     53 +/- 18 colonies per swatch                                      ______________________________________                                    

49% decrease in bacterial colonies by Endo-H treatment

B. For Staphylococcus epidermidis

    ______________________________________                                        B. For Staphylococcus epidermidis                                             ______________________________________                                        Control   57 +/- 11 colonies per swatch                                       Endo-H    16 +/- 10 colonies per swatch                                       ______________________________________                                    

72% decrease in bacterial colonies by Endo-H treatment.

These results indicate that Endo-H treatment significantly reduces thenumber of bacteria adhered to a cloth surface.

EXAMPLE 8 Binding of Endo-H to Bacteria

The following experiment was conducted to determine if the Type IIendoglycosidase, Endo-H, interacts with a surface component on thebacteria Staphylococcus aureus and Streptococcus mutans. Such aninteraction was detected. Although not completely characterized herein,this interaction was not previously known and may form the basis of theabove described ability of Endo-H to remove such bacteria from asurface.

Endo-H from transformed E. coli and purified by modifying the methodsdescribed by Trimble R. J. et al. (1985), J. Biol. Chem., 260,5638-5690, was labelled with biotin according to the procedure describedby Updyke, T. V. and Nicolson, G. L. (1986), Methods in Enzymology, 121,717-725. After such labelling, the Endo-H retained most of itsreactivity with the glycoprotein ovalbumin.

Overnight cultures of Staphylococcus aureus (ATCC 6538) grown in Luria'sbroth, and Streptococcus mutans (ATCC 27607) grown in Difco Brain HeartInfusion media, were centrifuged and washed three times with 200 mMNaCitrate pH 5.5 buffer and suspended in the same buffer to aconcentration of about 10⁹ cells/mi. Aliquots of 0.5 ml were placed in31.5 ml Eppendorf tubes and incubated under various conditions andtimes.

    ______________________________________                                                                               Incu-                                                         Biotiny-                                                                              0.2 M   bation                                                        lated   NaCitrate                                                                             Time                                   Tube Cells    2% BSA   Endo-H  pH 5.5  (min.)                                 ______________________________________                                        1    0.5 ml   5 μl  --      0.5 ml  30                                     2    0.5 ml   --       5 μl 0.5 ml   2                                     3    0.5 ml   --       5 μl 0.5 ml  30                                     ______________________________________                                    

Incubation was done at room temperature using a slow speed rocker foreither two or 30 minutes. BSA (bovine serum albumin), diluted intris-buffered saline was used as a control solution in order to preventany non-specific protein binding to the cells. After incubation, thetubes were centrifuged and the supernatants were discarded. Two cellwashes with 2% BSA solution were done by adding 1.0 ml BSA to the cells,vortexing well, centrifuging and discarding the supernatant. To thewashed cells, 0.5 ml of streptavidin-HRP (streptavidin-labeled horseradish peroxidase, Kirkegaard and Perry Laboratories, Inc.) was used andincubated for 30 minutes at room temperature. The tubes were againcentrifuged and washed as previously described. Detection of Endo-Hbinding to the bacterial cells was determined by the detection ofHRP-streptavidin, which will bind very tightly to the biotinylatedEndo-H bound to the cells. HRP detection was determined by adding 0.5 mlof the HRP substrate OPD (O-phenylenediamine) diluted in citratephosphate buffer solution containing hydrogen peroxide. The chromogengeneration was quenched with 2M H₂ SO₄ one minute after adding OPD. Thecells were centrifuged and the supernatant was read at 490 nm.

The following results were obtained:

    ______________________________________                                                       OD 490 nm                                                      ______________________________________                                        For Staphylococcus Aureus                                                     Control          0.13                                                         Endo-H, 2 minutes                                                                              1.89                                                         Endo-H, 30 minutes                                                                             1.90                                                         For Streptococcus mutans                                                      Control          0.18                                                         Endo-H, 2 minutes                                                                              3.76                                                         Endo-H, 30 minutes                                                                             3.80                                                         ______________________________________                                    

These results indicate that there is binding of Endo-H to the bacteriaStaphylococcus aureus and Streptococcus mutans. The data show that themajority of Endo-H that binds occurs in the first two minutes or lessafter contact with the cells. The higher absorbance obtained withStreptococcus mutans may indicate a higher level of Endo-H binding.

EXAMPLE 9

A heavy duty liquid laundry detergent composition of the presentinvention is as follows:

    ______________________________________                                                                 Active                                               Component                Weight %                                             ______________________________________                                        C.sub.13 linear alkylbenzene sulfonic acid                                                             8.00                                                 C.sub.14-15 alkyl polyethoxylate (2.25)                                                                12.00                                                sulfonic acid                                                                 1,2 propanediol          3.50                                                 Sodium diethylenetriamine pentaacetate                                                                 0.30                                                 Monoethanolamine         2.00                                                 C.sub.12-13 alcohol polyethoxylate (6.5)*                                                              5.00                                                 Ethanol                  8.50                                                 Potassium hydroxide      1.80                                                 Sodium hydroxide         3.85                                                 C.sub.12-14 fatty acid   10.00                                                Citric acid              4.00                                                 Calcium formate          0.12                                                 Sodium formate           0.86                                                 C.sub.12 alkyltrimethylammonium chloride                                                               0.50                                                 Tetraethylene pentamine ethoxylate (15-18)                                                             2.00                                                 Water                    35.12                                                Dye                      0.08                                                 Perfume                  0.25                                                 Protease**               0.125                                                Endoglycosidase H        2000 ppm                                             ______________________________________                                         Notes                                                                         (*) Alcohol and monoethoxylated alcohol removed.                              (**) mg active enzyme/g (@34 mg active enzyme/g stock)                   

The ingredients listed above are added to a mixing tank with a singleagitator in the order in which they appear. Before the protease enzyme,dye and perfume are added, the pH of the mix is adjusted so that a 10%by weight solution in water at 20° C. has a pH of about 8.5.

This composition provides superior cleaning of carbohydrate-containingstains, even compared to protease-containing and/or amylase-containingdetergents.

EXAMPLE 10

A heavy duty liquid laundry detergent composition of the presentinvention is as follows:

    ______________________________________                                                                 Active                                               Component                Weight %                                             ______________________________________                                        C.sub.13 linear alkylbenzene sulfonic acid                                                             8.00                                                 C.sub.14-15 alkyl polyethoxylate (2.25)                                                                12.00                                                sulfonic acid                                                                 1,2 Propanediol          3.50                                                 Sodium diethylenetriamine pentaacetate                                                                 0.30                                                 Monoethanolamine         2.00                                                 C.sub.12-13 alcohol polyethoxylate (6.5)*                                                              5.00                                                 Ethanol                  8.50                                                 Potassium hydroxide      1.80                                                 Sodium hydroxide         3.85                                                 C.sub.12-14 fatty acid   10.00                                                Citric acid              4.00                                                 Calcium formate          0.12                                                 Sodium formate           0.86                                                 C.sub.12 alkyltrimethylammonium chloride                                                               0.50                                                 Tetraethylene pentamine ethoxylate (15-18)                                                             2.00                                                 Water                    37.12                                                Dye                      0.08                                                 Perfume                  0.25                                                 Protease**               0.125                                                Endoglycosidase H        125 ppm                                              ______________________________________                                         Notes                                                                         (*) Alcohol and monoethoxylated alcohol removed.                              (**) mg active enzyme/g (@34 mg active enzyme/g stock)                   

The ingredients listed above are added to a mixing tank with a singleagitator in the order in which they appear. Before the protease enzyme,dye and perfume are added, the pH of the mix is adjusted so that a 10%by weight solution in water at 20° C. has a pH of about 8.5.

This composition provides superior cleaning of carbohydrate-containingstains, particularly fecal stains.

Other compositions of the present invention are obtained when the Endo Hlevel is reduced to 0.40 mg/ml, water is decreased to 35.72, and 1%Irgasan (a Ciba-Geigy antibacterial) is added.

EXAMPLE 11

A liquid soap composition of the present invention is as follows:

    ______________________________________                                                             Active                                                   Component            Weight %                                                 ______________________________________                                        Ammonium lauryl sulfate                                                                            6.0                                                      Sodium lauryl sarcosinate                                                                          5.7                                                      Cocamidopropyl betaine                                                                             6.3                                                      Coconut fatty acid   1.0                                                      Quaternary amine     0.3                                                      Ethylenediamine tetraacetic acid                                                                   0.2                                                      Ammonium sulfate     0.4                                                      Perfume              0.25                                                     Kathon               5 ppm                                                    Water                72.0                                                     Endoglycosidase H    1000 ppm                                                 Triclocarban         1.50                                                     ______________________________________                                    

The ingredients listed above are added to a mixing tank with a singleagitator in the order in which they appear below.

This composition provides antibacterial action for removal of commonskin flora, even when compared to non-glycosidase containing,antibacterial soaps.

EXAMPLE 12

A hard surface scouring cleanser of the present invention is as follows:

    ______________________________________                                        Component              Weight %                                               ______________________________________                                        False Body Fluid Phase 93.5                                                   (Specific Gravity 1.1)                                                        Barasum NAS-100        4.25                                                   (Sodium saponite clay)                                                        Tetrapotassium pyrophosphate                                                                         6.00                                                   Tripotassium phosphate 2.00                                                   Sodium hypochlorite bleach                                                                           0.90                                                   Sodium lauryl alkyl sulfate                                                                          0.25                                                   Surfactant                                                                    Dye and Perfume        0.26                                                   Endoglycosidase H      1000 ppm                                               Soft Water             78.86                                                  Abrasive               5.0                                                    (Expanded Perlite-Specific Gravity 2.0                                        Average Particle Diameter 50 microns)                                         Hercoflat 135 Filler   1.50                                                   (powdered polypro-pylene,                                                     Specific Gravity 0.9                                                          Average Particle Diameter 35 microns)                                         Ratio Average Particle                                                        Diameter Abrasive/Filler = 1.43:1                                             ______________________________________                                    

The composition is prepared by mixing tetrapotassium pyrophosphate,tripotassium phosphate, sodium saponite clay, dye, perfume and deionizedwater using relatively high shear agitation to the extent necessary toform a false body fluid phase. The alkyl sulfate surfactant is thenblended into this mixture followed by the polypropylene filler material.A separate aqueous slurry of sodium hypochlorite and perlite abrasive isprepared and then blended into the false body fluid phase while it isbeing liquified under moderate shear agitation. The resulting scouringcomposition is false bodied, i.e., gel-like in its quiescent state buteasily fluidized by application of shear stress. Such a composition isespecially effective for removal of stains and soil from hard surfaces.

EXAMPLE 13

A shampoo composition of the present invention is as follows:

    ______________________________________                                        Component           Level                                                     ______________________________________                                        Ammonium alkyl sulfate                                                                            55.25%                                                    (29% Aqueous solution)                                                        Zinc pyridinethione crystals of                                                                   2.0                                                       Ex. I of U.S. Pat. No. 4,345,080                                              Coconut monoethanolamide                                                                          3.0                                                       Ethylene glycol distearate                                                                        5.0                                                       Sodium citrate      0.5                                                       Citric acid         0.2                                                       Color solution      0.1                                                       Perfume             0.5                                                       Endoglycosidase H   1000 ppm                                                  Water               q.s. 100.00%                                              ______________________________________                                    

EXAMPLE 14

An antiperspirant stick of the present invention is made utilizing thefollowing components:

    ______________________________________                                        Component              Level                                                  ______________________________________                                        Cyclomethicone         42.55                                                  Fluid AP               4.99                                                   Stearyl alcohol        11.49                                                  Castor wax             4.99                                                   Talc                   6.99                                                   Zirconium/aluminum/glycine complex                                                                   26.67                                                  Fragrance masking agent                                                                              0.80                                                   C.sub.20 alcohol       0.12                                                   Pyridoxal phosphate    1.00                                                   Endoglycosidase H      500 ppm                                                ______________________________________                                    

EXAMPLE 15

A liquid soap composition of the present invention is as follows:

    ______________________________________                                                               Active                                                 Component              Weight %                                               ______________________________________                                        Ammonium lauryl sulfate                                                                              6.0                                                    Sodium alkyl sarcosinate                                                                             5.7                                                    Cocamidopropyl betaine 6.3                                                    Coconut fatty acid     1.0                                                    Ethylenediamine tetraacetic acid                                                                     0.2                                                    Ammonium sulfate       0.4                                                    Perfume                0.25                                                   Dye                     5 ppm                                                 Water                  80.15                                                  Endo-H                  50 ppm                                                2,4,4'-trichloro-2'-hydroxydiphenyl ether                                                            100 ppm                                                ______________________________________                                    

The ingredients listed above are added to a mixing tank with a singleagitator in the order in which they appear above. Before the dye andperfume are added, the pH of the mix is adjusted so that a 10% by weightsolution in water at 20° C. has a pH of about 6.5.

This composition provides antibacterial action for the removal of commonskin flora.

EXAMPLE 16

A hard surface cleanser of the present invention is as follows:

    ______________________________________                                                           Active                                                     Component          Weight %                                                   ______________________________________                                        Sodium lauryl alkyl sulfate                                                                      0.5                                                        Sodium alkyl sulfate                                                                             0.5                                                        Butyl carbitol     4.0                                                        Sodium bicarbonate 0.5                                                        Citric acid        0.04                                                       Formaldehyde       0.03                                                       Perfume            0.05                                                       Tartrate mono/disuccinate                                                                        5.0                                                        Endo-H             1000 ppm                                                   Water              88.4                                                       ______________________________________                                    

The ingredients listed above are added to a mixing tank with a singleagitator in the order in which they appear above. Before the perfume isadded, the pH of the mix is adjusted so that a 10% by weight solution inwater at 20° C. has a pH of about 7.

This composition is effective for the removal of soap scum and mold fromhard surfaces, and is more efficacious than a cleanser without theendoglycosidase.

EXAMPLE 17

A composition used for the cleaning and/or preservation of whole fruit,vegetables or other plant surfaces is as follows:

    ______________________________________                                                              Active                                                  Component             Weight %                                                ______________________________________                                        Water                 96.4                                                    C.sub.12-13 alcohol polyethoxylate (6.5)                                                            0.1                                                     Endo-H                3500 ppm                                                ______________________________________                                    

This composition is prepared by mixing the alcohol polyethoxylate andEndo-H in water at their respective levels and adjusting the final pH tobetween 6-7. The final composition, when sprayed on plant surfaces suchas whole fruit or vegetables, is useful in preventing microbial growthon said surfaces.

EXAMPLE 18 Potentiation of Bacteriocidal Effect of Antimicrobial byEndo-H

An overnight culture of Escherichia coli was diluted into fresh nutrientbroth and grown for four hours at 37° C. Cells were obtained bycentrifugation and washed in 0.2M Na-citrate buffer (SCB) pH 5.5. Aftercentrifuging, cells were resuspended in SCB. The following tubes (induplicate) were prepared:

    ______________________________________                                                  1000 ppm  5000 pm                                                   Condition Endo-H    Chlorhexidine                                                                            SCB    Water                                   ______________________________________                                        Control    0 μl   0 μl   200 μl                                                                            10 μl                                Chlorhexidine                                                                            0 μl  10 μl   200 μl                                                                            10 μl                                Endo-H    200 μl  0 μl    0 μl                                                                             10 μl                                Endo-H    200 μl 10 μl    0 μl                                                                              0 μl                                  +                                                                           Chlorhexidine                                                                 ______________________________________                                    

The Endo-H was from E. coli producing S. plicatus Endo-H. To each tube,790 μl of cell suspension added (final volume now 1 ml) and 10 μlsamples were taken out as a 0 min control. Tubes were incubated at 37°C., on a rotary shaker and 10 μl samples were removed at 1 and 3 hours.The 10 μl aliquots were mixed with 990 μl of PBS (Phosphate bufferedsaline) (10⁻² dilution) and diluted further sequentially (1:10) in PBS(100 μl in 900 μl of PBS). 10 μl of each diluted solution was plated onLuria-Bertani agar plates. The plates were incubated at 37° C. overnightand colonies were counted. Number of colony forming bacteria in tubeswere calculated according to dilutions made and the logarithm of thisnumber used for further graphs and calculations.

    ______________________________________                                                   0 minute   1 hour    3 hours                                       Condition  Control    (log kill)                                                                              (log kill)                                    ______________________________________                                        Control    8.62       8.57 (.05)                                                                              8.53 (.09)                                    200 ppm    8.64       8.55 (.09)                                                                              8.55 (.09)                                    Endo-H                                                                        50 ppm     8.60       4.42 (4.15)                                                                             2.44 (6.59)                                   Chlorhexidine                                                                 200 ppm    8.61       2.40 (6.17)                                                                             2.00 (>6.53)                                  Endo-H +                                                                      Chlorhexidine                                                                 ______________________________________                                    

These results are plotted in FIG. 8. As can be seen, 200 ppm Endo-Henhances the bacteriocidal effect of 50 ppm chlorhexidine.

Similar results were obtained for slightly different concentrations ofchlorhexidine and Endo-H as measured over a one hour time period. Theseresults are depicted in FIG. 9. As can be seen, 140 ppm of Endo-Henhances the efficacy of 40 ppm chlorhexidine.

To further investigate this effect, a similar experiment was conductedusing 20 ppm chlorhexidine (final concentration) with varyingconcentrations of Endo-H. The results are shown in FIGS. 10A and 10B.These plots represent the change in the log of colony forming units(CFU). As can be seen, a relatively linear relationship exists betweenthe amount of Endo-H added through about 280 ppm Endo-H. Furtherincreases in Endo-H concentration enhance the adverse effect onbacterial viability through at least 1000 ppm Endo-H in combination with20 ppm chlorhexidine.

EXAMPLE 19 Effect of Endo-H Alone and in Combination with Antimicrobialon Viability of Fungi

A log phase culture of Candida albicans was grown, diluted into freshgrowth medium, and treated with 0, 1, 10, 100 and 1000 ppm Endo-H (finalconcentration) for 4 hours while incubating with agitation at 37° C. TheEndo-H was from Bacillus subtilis transformed to produce Endo-H from S.plicatus. One, ten and one hundred fold dilutions were made and platedto give viable cell counts. Zero through 10 ppm Endo-H did notsignificantly reduce cell viability, although in one case 10 ppm Endo-Hreduced viability by about 36% after 18 hours of incubation. However,100 ppm to 1000 ppm Endo-H reduced the number of viable cells recoveredby about 50% to 88%, respectively, compared to the control not treatedwith Endo-H when treated for four hours.

In a separate experiment, a culture of Candida albicans was grown,diluted into fresh medium, and treated with 2.5 μg/ml Nystatin® inaddition to either 0, 1, 10, 100 or 1000 ppm Endo-H (finalconcentration) for 18 hours, while incubating with agitation at 37° C.One, ten, one hundred and one thousand fold dilutions were made andplated to give viable cell counts. Endo-H reduced viable cells recoveredas follows as compared to that obtained with Nystatin® alone:

    ______________________________________                                        ppm Endo-H     % Reduction                                                    ______________________________________                                        0                   0%                                                        1          ppm     69%                                                        10         ppm     93%                                                        100        ppm     99%                                                        ______________________________________                                    

As can be seen, as little as 1 ppm Endo-H significantly enhances themycocidal effect of Nystatin® whereas 10 ppm and 100 ppm Endo-H killalmost all of the fungi surviving Nystatin® treatment alone.

A similar experiment was conducted using Amphotericin B® at aconcentration of 0.5 micrograms per ml for three hours. The results wereas follows:

    ______________________________________                                        ppm Endo-H   % reduction in viability                                         ______________________________________                                        0            0                                                                1            17%                                                              10            5%                                                              100          96%                                                              1000         94%                                                              ______________________________________                                    

As can be seen, 100 ppm of Endo-H enhances the mycocidal effect ofAmphotericin B®.

EXAMPLE 20 Antimicrobial Effect of Endo-H Alone or in Combination with aLysozyme

A 48-hour subculture of E. coli (ATCC 31617) was used to test the effectof the lysozyme mutanolysin (Sigma Chemical Co.) alone or in combinationwith detergent and/or Endo-H. The Endo-H was from E. coli transformed toproduce Endo-H from S. plicatus. The following protocol and results wereobtained after treatment for two hours at 37° C.:

    ______________________________________                                                 Na Citrate                                                           Mutano-  pH        Tide     Endo-H                                            lysin    5.5    7.0    200 ppm                                                                              200 ppm                                                                              Results                                  ______________________________________                                        1   Control                            Fimbriae, tight                                                               cell wall                              2   200 ppm  +                         Fimbriae, tight                                                               cell wall                              3   200 ppm         +                  Fimbriae, tight                                                               cell wall                              4   200 ppm  +           +             Fimbriae, tight                                                               cell wall                              5   200 ppm         +    +             Fimbriae, cell                                                                condensation                           6   200 ppm  +                  +      Loss of fimbriae                       7   200 ppm         +           +      Few cells, some                                                               ghosts, cell wall                                                             disintegration                         8   200 ppm  +           +      +      Some fimbriae                          9   200 ppm         +    +      +      Cells in bad                                                                  shape (con-                                                                   densed) but                                                                   still present                          ______________________________________                                    

As can be seen, the gross morphology of the bacteria exposed to Endo-Hand mutanolysin either with or without detergent at various pH, wassignificantly modified. The most dramatic effects occurred at pH 7 whenEndo-H was used alone or in combination with detergent. Cell viability,however, was apparently not effected. Endo-H and mutanolysin did notreduce the number of colonies obtained in a plating experiment ascompared to a buffer control.

EXAMPLE 21 Bacterial Removal from Glass Surfaces by Endo-H and PNGase F

Escherichia coli (ATCC 31617) and Staphylococcus epidermidis (ATCC 155)were used to inoculate glass slides. Each slide contained two etchedcircles and each was inoculated with E. coli or S. epidermidis.

The bacteria were allowed to incubate at 37° C. for two hours.

After rinsing with distilled water, the slides were treated witheither 1) PBS buffer, 2) Endo-H (100 ppm) in PBS buffer, or 3) PNGase F(100 ppm) in PBS buffer. The Endo-H was derived from E. coli producingS. plicatus Endo-H. After 30 minutes at 37° C. the slides were rinsed indistilled water. After Gram staining, the slides were read with brightfield optics on a light microscope.

In the case of the buffer control, the number of bacteria remaining onthe slide was greater than 100 per field. The slides treated with Endo-Hcontained far fewer bacteria. In the case of S. epidermidis, only about1 to 3 bacteria were observed per field. In the case of E. coli, about 5to 10 were observed per field. For those slides treated with PNGase F,moderate numbers of bacteria were observed for both S. epidermidis andE. coli (approximately 20 per field).

These results indicated that PNGase F is capable of removing bacteriafrom glass surfaces albeit not as efficiently as Endo-H.

EXAMPLE 22 Tablet Denture Cleaner with Endo-H

Sodium bicarbonate, sodium perborate monohydrate, tartaric acid, sodiumtripolyphosphate, sulphamic acid, polyethylene glycol (20,000 m.wt.) andethylene diamine tetraacetate are separately granulated by fluidizing ina hot air bed at 60°-65° C. for 30 minutes. Such granulates are thentumble mixed with the other ingredients to produce a "first layer"mixture and a "second layer" mixture, wherein the "first layer" mixturehas the following composition:

    ______________________________________                                                            % by Weight                                               ______________________________________                                        Sodium bicarbonate    30.00                                                   Tartaric acid         23.00                                                   Potassium monopersulphate                                                                           16.00                                                   Sulphamic acid        11.00                                                   Oisodium pyrophosphate                                                                              8.20                                                    Sodium carbonate      3.90                                                    Polyethylene glycol   12.60                                                   Sodium sulphate       2.00                                                    Peppermint powder     2.50                                                    Silicon dioxide       1.30                                                    Sodium dodecyl benzene sulphonate                                                                   0.50                                                    ______________________________________                                    

and the "second layer" mixture has the following composition:

    ______________________________________                                                            % by Weight                                               ______________________________________                                        Sodium perborate monohydrate                                                                        30.00                                                   Potassium monopersulphate                                                                           28.00                                                   Sodium bicarbonate    13.34                                                   Sodium tripolyphosphate                                                                             10.00                                                   Sodium bicarbonate/colour                                                                           4.00                                                    Trilon B              3.00                                                    Sodium carbonate      3.00                                                    Polyethylene glycol   2.50                                                    Silicone dioxide      2.00                                                    Peppermint powder     1.50                                                    Wasag ester 7         0.70                                                    Wasag ester 15        0.70                                                    Hardened triglycerides                                                                              0.50                                                    Sodium dodecyl benzene sulphonate                                                                   0.40                                                    Succinate detergent   0.30                                                    Blue Lake No. 1       0.06                                                    Endo-H                100 ppm                                                 ______________________________________                                    

A tablet is produced by compressing in a HORN rotary tableting press of39 stations. Compressing is in two stages: Initially the "second layer",blue mixture is compressed to very low pressure (10 kN per tablet) byway of tamping. The "first layer", white mixture is then instilled andpressed to 70 kN per tablet. In this way a tablet of 4 grams is producedbeing 2.7 grams blue and 1.3 grams white.

Tablets are dissolved in water by the consumer to clean dentures placedin the water.

EXAMPLE 23 Light Cream with Endo-H

An oil-in-water sunscreen emulsion base is made from the followingingredients, which are indicated by their chemical or Cosmetic, Toiletryand Fragrance Association (CTFA) name:

    ______________________________________                                        Association (CTFA) name:                                                      Ingredient             Weight %                                               ______________________________________                                        Water Phase:                                                                  Methylparaben (preservative)                                                                         0.20                                                   Pantethine (moisturizer)                                                                             0.10                                                   Carbomer 934 (thickener)                                                                             0.08                                                   Sodium hydroxide, 10% (neutralizer)                                                                  1.00                                                   Endo-H                 100 ppm                                                Purified water, q.s. to                                                                              100%                                                   Oil Phase:                                                                    Heavy mineral oil      4.00                                                   Stearic acid, double pressed                                                                         3.00                                                   (anionic emulsifier)                                                          Cholesterol (auxiliary emulsifier)                                                                   1.00                                                   Cetyl alcohol (auxiliary emulsifier)                                                                 1.80                                                   Castor oil (emollient) 1.00                                                   Cetyl palmitate (emollient)                                                                          1.20                                                   Octyl dimethyl PABA (U.V.-absorber)                                                                  1.40                                                   Propylparaben (preservative)                                                                         0.10                                                   ______________________________________                                    

In a mixing vessel equipped with a mechanical stirrer, water and thewater phase ingredients other than the sodium hydroxide and Endo-Haqueous solution are added and mixed with heating to about 75°-80° C. toform a uniform aqueous dispersion. The sodium hydroxide solution is thenadded and mixed into the aqueous phase to neutralize the acidic Carbomerthickener.

In a separate mixing vessel, the mineral oil and oil phase ingredientsare added and mixed with heating to about 80°-82° C. to form a uniformoil phase. The heated oil phase is slowly added to the heated waterphase using high speed mechanical dispersing means. Mixing is continueduntil a homogeneous oil/water emulsion is obtained. The emulsion iscooled to room temperature. If desired, optional colorants such aswater-soluble dyes are preferably mixed into the emulsion at about45°-50° C. and fragrant oils are preferably added at about 35°-40° C.Endo-H is mixed into the emulsion at about 35°-40° C.

EXAMPLE 24 Removal of S. aureus from Pig Skin

Pig skin was inoculated with S. aureus (1.2×10⁷ colonies/ml) byspreading 0.1 cc of the culture on the skin surface. The organisms wereallowed to set on the skin for two hours at room temperature. Duplicatepieces of skin were then treated for 30 seconds with:

1) untreated control

2) water alone

3) 10% soap solution

4) #3+Endo-H (20 ppm)

5) 20 ppm Endo-H in buffer

The Endo-H was obtained from E. coli transformed to produce Endo-H fromS. plicatus. After treatment the samples were rinsed in distilled waterand placed in 2% osmium tetroxide followed by fixation inRyter-Kellenberger fixative. The samples were then processedalternatively in osmium and thiosemicarbizone. After critical pointdrying, all samples were examined on the SEM. Photomicrographs weretaken.

S. aureus colonies were found in abundance on the untreated, watertreated, or plain soap treated samples. See, e.g. FIG. 11 whichdemonstrates the effect of treatment with liquid hand soap. TheEndo-H-treated samples demonstrated a significant loss of organisms.See, e.g. FIG. 12 which demonstrates the removal of S. aureus from swineskin when treated with liquid hand soap plus Endo-H.

EXAMPLE 25 Mold Removal from Shower Curtain

A plastic shower curtain was moistened with tap water and placed in thedark for 3 weeks. At the end of that time, a small sample of the curtainthat was covered with mold was treated with:

1) distilled water

2) +2000 ppm Joy detergent

3) +1000 ppm Endo-H

4) untreated

The Endo-H was obtained from E. coli transformed to produce Endo-H fromS. plicatus. The treatments lasted 10-15 seconds at room temperature.The shower curtain was wiped off after treatment with a cotton swab.

FIG. 13 depicts the results obtained. The non-treated control (lowerright photograph--lower right quadrant of center photograph) showedabundant mold and mildew particles both macro and microscopically.

The distilled water control (upper right photograph--upper rightquadrant of center photograph) showed less organisms, although particlesstill remained and discoloration was evident.

The Joy-treated control (lower left photograph--lower left quadrant ofcenter photograph) showed less organisms than the water treated sample,but discoloration was still evident.

The Endo-H treated sample (upper left photograph--upper left quadrant ofcenter photograph) was free of both organisms and any discolorations.

EXAMPLE 26 Bacterial Removal from Fabric

Fabric swatches were cut to the size of a petri dish. Additional fabricwas added to reach a 5% fabric load (which was not inoculated). Theswatches were sterilized in an autoclave for 15 minutes at 15 lbs. 121°C. One fabric load is needed for each treatment. Glass beads (40 g) and100 mls 0.2M pH 7.0 citrate buffer was placed into 250 ml Erlenmeyerflasks. The flasks were plugged with rubber stoppers and aluminum foiland sterilized in an autoclave. E. coli subcultured into fresh nutrientbroth and allowed to incubate for 48 hours at 37° C. Half strengthtrypticase soy agar plates (10 g/500 mls) were prepared and sterilized.After cooling, tetrazolium (1 ml/liter) was added.

The agar plates were inoculated as follows:

1) serial dilutions from the 48-hour culture were prepared (1:10, and 10fold dilutions through three more tubes in peptone water);

2) Thereafter, each swatch was inoculated with 2 mls of the lastdilution (10⁴).

3) The swatches were then incubated at 37° C. for two hours (twoswatches/treatment).

After incubation, the swatches were laundered as follows:

Wash

100 mls sterile 0.2 M pH 7.0 citrate buffer+40 g glass beads+thetreatment described in FIG. 14 (where AWA is Endo-H from E. colitransformed to produce Endo-H from S. plicatus) in a 250 ml Erlenmeyerflask (sterile). Two inoculated swatches+sterile fabric to make 5%fabric load were washed at 95° F. for 12 minutes with shaking.

Rinse

After washing, the swatches were rinsed by adding 100 mls sterile doublydistilled/deionized water+40 g glass beads in a 250 ml Erlenmeyer flask(sterile) at room temperature for two minutes with shaking.

The fabric swatches were then placed in petri dishes and overlaid with 3mls of one-half strength trypticase soy agar with tetrazolium. Afterincubation for 48 hours, the colonies were counted.

The results are shown in FIG. 15. These results indicate that 2% Irgasanplus Liquid Tide provide a two log decrease in bacterial growth ascompared to Tide alone. The addition of 40 ppm Endo-H, however, reducesbacterial growth another log unit.

EXAMPLE 27 Effect of Endo-H on Yeast

Broth cultures (18 hour) of Candida albicans and Sacchromyces cerevisiaewere treated with:

1) 0.2M Na citrate buffer, pH 5.5

2) #1 plus 200 ppm Endo-H (from E. coli producing S. plicatus Endo-H)

The treatments lasted 2 hours at 37° C.

After treatment, an aliquot of each was placed on a formvar-coated 200mesh copper grid, and examined by TEM. Photomicrographs of theexaminations were taken and are presented in FIGS. 15 and 16.

As can be seen in FIG. 15A, Candida treated with buffer alone was ingood morphological condition. As indicated in FIG. 15B, Candida treatedwith Endo-H leaked material at a rapid rate and lost structuralintegrity.

Sacchromyces treated with buffer alone was in good morphologicalcondition as can be seen in FIG. 16A. When treated with Endo-H, however,all that remained were very limited pieces of membranous material. SeeFIG. 16B.

EXAMPLE 28 Effect of Endo-H and Lysozyme on Viability of E. Coli

A culture of E. coli K12 grown overnight in Laurie Broth (LB), wasdiluted 1:1000 in LB and regrown for 4 hours at 37° C. Cells werecentrifuged, washed and resuspended in 0-1M NA-acetate pH 5.5 (NA)buffer. Eight tubes were set up as follows:

    ______________________________________                                                      Tube Number                                                                   1,2  3,4      5,6    7,8                                        ______________________________________                                        μl cells     800    800      800  800                                      μl NA buffer 200    --       --   200                                      μl Endo-H (1 mg/ml)                                                                        --     200      200  --                                       ______________________________________                                    

The Endo-H was from B. subtilis transformed to produce Endo-H from S.plicatus. Tubes were incubated for one hour at 37° C. Tubes werecentrifuged, washed and resuspended in 8.00 μl of 0.1M Na-phosphate, pH7.2 (NP) buffer containing 0.1M EDTA. Buffer or hen egg white lysozymesolution was added to tubes as follows:

    ______________________________________                                                       Tube Number                                                                   1,2  34,      56,    7,8                                       ______________________________________                                        μl NP buffer  200    200                                                   μl lysozyme (1 mg/ml)                                                                       --     --       200  200                                     ______________________________________                                    

Aliquots were taken at this time to determine colony forming units (CFU)(Column A). After incubation for one hour at 37° C. aliquots were usedto determine CFUs (Column B). The log of colony forming units werecalculated. The decrease in log CFUs was determined by subtracting Bfrom A. The results are shown below:

    ______________________________________                                                      Log CFUs      Change                                            Condition       A      B        in log CFUs                                   ______________________________________                                        Control         7.89   7.90     +0.01                                         Endo-H (200 ppm)                                                                              8.21   7.92     -0.29                                         Lysozyme (200 ppm)                                                                            7.87   7.68     -0.19                                         Endo-H + lysozyme                                                                             8.17   7.53     -0.64                                         ______________________________________                                    

These results indicate that the combination of Endo-H and lysozymedecreases the viability of E. coli as compared to Endo-H or lysozymealone.

EXAMPLE 29 Comparison of Endo-H with T-4 or Hen Egg White Lysozyme onViability of E. coli

E. coli cells were washed and suspended in 0.1M Na-acetate pH 5.5buffer. Cells were aliquoted (10 ml) in two tubes. To one tube, onlybuffer was added (control) and to another Endo-H was added (treated).The Endo-H was from B. subtilis transformed to produce Endo-H from S.plicatus. Cells were incubated for one hour at 37° C. Cells werecentrifuged, washed and resuspended in 0.1M Na-phosphate (pH 7.2)buffer. Cells were aliquoted equally and incubated either with buffer orlysozyme. Hen egg white (HL) and T4 (TL) lysozymes were compared in thisexperiment. Tubes were incubated for 1.5 hours. Samples were diluted andplated for CFU determination before (A) and after (B) incubation. Thelog of CFUs were determined. The following results were obtained.

    ______________________________________                                        Incubation Condition Log CFUs  Change in                                      First       Second       A      B    log CFUs                                 ______________________________________                                        Endo-H (300 ppm)                                                                            --         7.60   7.23 -0.37                                      --        HEWL (445 ppm)                                                                             6.69   6.73 -0.23                                    Endo-H (300 ppm)                                                                          HEWL (445 ppm)                                                                             7.41   6.81 -0.60                                      --        TL (445 ppm) 4.98   4.53 -0.45                                    Endo-H (300 ppm)                                                                          TL (445 ppm) 5.27   4.30 -0.93                                    ______________________________________                                    

These results indicate that T-4 lysozyme is also effective in reducingthe viability of E. coli in combination with Endo-H.

EXAMPLE 30 Treatment of Soiled Diaper Material with Endo-H

Samples were obtained from a soiled diaper. Each sample was divided. Theleft side of the sample was washed in 2000 ppm Tide and 1 ppm BPN'(subtilisin protease from Bacillus amyloliquifaciens). The right sidewas washed in 2000 ppm Tide, 1 ppm BPN' and 40 ppm Endo-H (BoehringerMannheim Biochemical Catalog No. 100 119). Each sample was washed for 12minutes at 95° F. The results of two experiments are shown in FIGS. 17and 18. As can be seen, the diaper material on the right side of FIGS.17 and 18 contains substantially less fecal stain as compared to theTide-protease treated diaper shown on the left of FIGS. 17 and 18.

Having described the preferred embodiments of the present invention, itwill appear to those of ordinary skill in the art that variousmodifications may be made and that such modifications are intended to bewithin the scope of the present invention. Other compositions of thepresent invention are obtained when Endo-D or F or PNGase F aresubstituted for Endo-H in the Examples.

All references cited herein are expressly incorporated by reference.

What is claimed is:
 1. A cleaning composition comprising:a) a firstenzyme and a second enzyme, wherein said first enzyme is a Type IIendoglycosidase selected from the group consisting of Endo-D, Endo-H,Endo-L, Endo-C, Endo-CII, Endo-F-Gal type, Endo-F and PNGaseF, and saidsecond enzyme is selected from the group consisting of proteases,lipases, nucleases, glycosidases different from said first enzyme, andcombinations thereof; b) a detergent surfactant; and c) a detergentbuilder.
 2. The composition of claim 1 wherein said Type IIendoglycosidase is Endo-H.
 3. A cleaning composition of claim 1 whereinthe Type II endoglycosidase is present in an amount effective forremoval of glycosidase-containing substances from surfaces.
 4. Thecleaning composition of claim 1 wherein said second enzyme is aprotease.
 5. The cleaning composition of claim 1 wherein said firstenzyme is Endo-H and said second enzyme is a protease.
 6. The cleaningcomposition of claim 5 wherein said second enzyme is a subtilisin or amutant subtilisin.
 7. The composition of claim 1 wherein said differentglycosidase is a Type II endoglycosidase.
 8. The composition of claim 1wherein said different glycosidase is an exoglycosidase.
 9. Thecomposition of claim 1 further comprising a surfactant selected from thegroup consisting of anionic, nonionic, ampholytic, zwitterionic andcationic surfactants.
 10. The composition of claim 1 further comprisinga disulfide cleaving reagent.
 11. The composition of claim 1 whereinsaid second enzyme is a subtilisin or mutant subtilisin.
 12. A cleaningcomposition according to claim 3 having a pH of from 4 to
 10. 13. Acleaning composition according to claim 3 comprising a Type IIendoglycosidase selected from the group consisting of Endo-H, Endo-F andEndo-D and having a pH of from 5 to
 8. 14. A cleaning compositionaccording to claim 3 further comprising a protease enzyme.
 15. Acleaning composition according to claim 13 further comprising from about1% to 90% by weight of detergent surfactant.
 16. A cleaning compositionaccording to claim 15 comprising from about 5% to 50% by weight ofdetergent surfactant.
 17. A cleaning composition according to claim 3further comprising from about 1% to 75% by weight of detergent builder.18. A cleaning composition according to claim 17 comprising Type IIendoglycosidase selected from the group consisting of Endo-H, Endo-F andEndo-D, and mixtures thereof, having a pH of from 6 to
 10. 19. Acleaning composition according to claim 18 comprising from about 5% toabout 40% by weight of detergent builder selected from the groupconsisting of fatty acids, polycarboxylates, polyphosphates, andmixtures thereof.
 20. A cleaning composition according to claim 19formulated as a liquid comprising from about 10% to about 40% by weightof detergent surfactant, and from about 10% to about 20% by weight ofdetergent builder, and from about 20 ppm to 200 ppm of Endo-H, Endo-F orEndo-D.